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 and withdrawal of claims 19 and 20 in the reply filed on 4/22/26 is acknowledged.
Claim(s) 1-20 is/are currently pending. Claim(s) 1-18 are herein examined on their merits.
Priority
Claim to benefit of provisional application 63/376,911 is acknowledged and the effective filing date of the instant application is considered to be September 23, 2022.
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 1-18 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.
Regarding claims 1-18, the phrase “e.g.”/"for example" recited in claim 1 renders the claims indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claims 1-18, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 1 recites the broad recitations “at least 25%” and “or at least 10”, and the claim also recites “at least 50%” and “at least 15, 20, 25, or more” which are the narrower statements of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claims 14-16 are rejected for the recitation of “the 5’ universal sequence” because it is unclear whether this sequence is required to be at the 5’ end of the appropriate primer AND the probe or if it is only required to be at the 5’ end of the appropriate primer. Clarification is requested.
Claim 17 is rejected for the recitations of “or a mutant thereof,” as being indefinite. A clear limiting definition is not given in either the claims or the specification. The term “mutant” may be broadly interpreted to mean an enzyme which differs in amino acid sequence at one or more residues compared to a reference. Therefore, for example, it is unclear if the language of claim 17 encompasses all of the following: human-engineered enzymes which differ from a reference enzyme (e.g. single residue mutants, mutants lacking certain domains, etc.); enzymes from the same species with <1% difference in sequence from a reference; enzymes from closely related species which differ by 10% or more in sequence from a reference. Therefore, a person with ordinary skill in the art would not be reasonably apprised of the scope of the invention.
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1, 2, 5, 11, and 18 are rejected under 35 U.S.C. 103 as unpatentable over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), and as evidenced by Wikipedia (published Oct. 23 2004; Wikimedia Foundation, Inc. “Klenow fragment”. Retrieved from: https://web.archive.org/web/20041023033327/http://en.wikipedia.org/wiki/Klenow_fragment), Pohl et al (published Jan. 2004; Pohl et al. Expert Rev Mol Diagn. 2004 Jan;4(1):41-7), and Wangh et al. (published March 18, 2004; Patent Application Publication US-20040053254-A1).
Rajagopal recites a method of detecting a target nucleic acid in a sample (Abstract).
Regarding claim 1, Rajagopal the method comprising forming a reaction mixture comprising: sample nucleic acids (par 87-88); a plurality of forward primers comprising 3' target-specific forward sequences, a plurality of reverse primers comprising 3' target-specific reverse sequences (par. 91-93), wherein the forward primers or the reverse primers further comprise a 5' universal sequence (par. 54); probe nucleic acids comprising: a fluorophore and a quencher (par. 109-110), and 100% of the 5' universal sequence (par. 107); and a DNA polymerase and an RNaseH2 enzyme (par. 85-86).
Rajagopal recites annealing the forward primers to target nucleic acids in the sample nucleic acids and extending with a polymerase the forward primers using the target nucleic acids as a template to form first strand extension products; annealing the reverse primers to the first strand extension products and extending with the polymerase the reverse primers using the first strand extension products as a template to form second strand extension products, wherein the second strand extension products comprise a reverse complement of the 5' universal sequence if the forward primers comprise the 5' universal sequence (par. 57; Fig. 5AB); and annealing the probe nucleic acids to the reverse complement of the 5'universal end sequence wherein an enzyme cleaves the annealed probe, thereby separating the quencher from the fluorophore to generate a detectable signal indicating the presence of the target nucleic acid (par 117; Fig. 5AB). In this scenario, the forward primer is the ‘tailed primer’; the reverse primer is the ‘target specific reverse primer’; the probe nucleic acid is the ‘universal probe’; and the 5’ universal sequence is the ‘universal probe binding motif.’ The probe binds to the antisense of the universal sequence. See below.
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Note that regarding the universal sequence, Rajagopal recites that “a probe may be complementary or homologous to a universal probe binding motif,” which is interpreted to mean fully, or 100%, complementary or homologous to the universal sequence. This meets the limitation that at least 25% of the 5’ universal sequence be comprised in the probe nucleic acid.
Note that the choice of which strand extension product contains the reverse complement of the 5’ universal sequence is arbitrary. If samples contain dsDNA (as encompassed by the claim), the reverse complement of the 5’ universal sequence will be on both first and second strand extension products, regardless of which primer contains the sequence. In the same reaction, target nucleic acids will be separated into sense and antisense strands and both the forward and reverse primers may simultaneously generate first strand extension products on separate molecules. If the 5’ universal sequence is comprised on the forward primer, reactions will be occurring simultaneously wherein the reverse complement of the 5’ universal sequence is comprised on both the first and second strand extension products. As Rajagopal also encompasses dsDNA (par. 44, 80), and because the terms “forward” and “reverse” with regard to primers are perspective-dependent, the limitation that wherein the first strand extension products comprise a reverse complement of the 5' universal sequence if the reverse primers comprise the 5' universal sequence is also considered to have been met.
Regarding claim 2, Rajagopal recites that the method occurs in partitions wherein the target nucleic acid is distributed among the partitions such that at least a portion of the partitions do not contain a target nucleic acid (par 75). Digital PCR by definition requires that at least some partitions do not contain nucleic acid templates, as evidenced by Pohl (pg. 41, col. 1, 2nd par.).
Regarding claim 5, Rajagopal recites that the partitions may be droplets (par. 87).
Regarding claim 11, Rajagopal recites TaqMan® probes (par. 50). As evidenced by Wangh, TaqMan® probes are linear probes (Wangh: par. 6).
Regarding claim 18, Rajagopal recites the use of DNA polymerases which lack 5'-3' exonuclease activity (par. 86). For example, as evidenced by Wikipedia, the Klenow fragment lacks 5’-3’ exonuclease activity (Wikipedia: “Klenow fragment”).
Regarding claim 1, Rajagopal does not recite that the probe comprises at least one ribonucleotide separating the fluorophore and the quencher, nor that the RNase H2 enzyme cleaves the annealed probe at the ribonucleotide, separating the quencher from the fluorophore to generate a detectable signal indicating the presence of a target nucleic acid.
Regarding claim 1, Liu recites the use of a molecular beacon probe for SNP detection which includes at least one ribonucleotide (Abstract). The ribonucleotide is recited as separating the fluorophore and the quencher (Fig. 7). Liu recites that the RNase H2 enzyme cleaves the annealed probe at the ribonucleotide, thereby separating the quencher from the fluorophore to generate a detectable signal indicating the presence of a target nucleic acid (Abstract; Fig. 7).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu. One would have been motivated to do so in order to use a relatively cheap fluorescence-labeled probe which does not require a subsequent step of gel electrophoresis compared to other methods of genotyping (pg. 91, col. 2, 3rd full par.)
Claims 3, 4, and 6 are rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claims 1 and 2 above, and further in view of Tong et al. (published Oct 18, 2017; Tong et al. Cellular Physiology and Biochemistry (2017) 43 (4): 1718–1730). Note that claim 1 was evidenced by Pohl and Wikipedia.
Rajagopal and Liu teach the limitations of claims 1 and 2, as discussed above.
Regarding claims 3 and 4, neither Rajagopal nor Liu explicitly teach that sample nucleic acids are cell-free DNA, nor that they’re from a pregnant woman and contain maternal and fetal DNA. Regarding claim 6, neither recites that the partitions are microwells.
Regarding claims 3 and 4, Tong teaches detecting sample nucleic acids which are cell-free DNA and which are from a pregnant woman and contain maternal and fetal DNA (pg. 1724: Prenatal diagnosis of genetic disease).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Tong. One would have been motivated to do so in order to avoid the use of invasive sampling techniques and their associated risks (pg. 1724: Prenatal diagnosis of genetic disease).
Regarding claim 6, Tong teaches microwell partitions (pg. 1719: Digital PCR). Although Rajagopal does not teach microwells explicitly, Rajagopal does teach digital PCR (which requires partitioning of samples) and the use of droplets, as discussed for claim 2.
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Tong. One would have been motivated to do so because Tong teaches that droplets and microwells are among the few techniques for partitioning of samples in dPCR, and are therefore functional equivalents (pg. 1719: Digital PCR).
Claims 7 and 8 are rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claim 1 above, and further in view of Poddar (published February 2000 ; Mol Cell Probes. 2000 Feb;14(1):25-32). Note that claim 1 was evidenced by Pohl and Wikipedia.
Rajagopal and Liu teach the limitations of claim 1, as discussed above.
Regarding claims 7 and 8, Rajagopal and Liu do not explicitly teach any relative concentrations of the forward and reverse primers.
Poddar teaches antisense primers having a higher concentration than antisense primers in PCR to generate more single-stranded sense DNA fragments which are the target of molecular beacon probes (pg. 28, col. 2).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Poddar. One would have been motivated to do so because asymmetric PCR can provide an improved level of signal and/or higher sensitivity of detection (pg. 30, col 2). In a molecular probe scenario, the strand complementary to the target strand will compete with the probe for hybridization to the target; therefore, it would be logical to increase the concentration of whichever primer produces the target strand which binds the probe (pg. 26, col. 1).
Claims 9 and 10 are rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claim 1 above, and further in view of Afonina et al (published Dec 2007; Afonina et al. Biotechniques. 2007 Dec;43(6):770, 772, 774).
Rajagopal and Liu teach the limitations of claim 1, as discussed above.
Regarding claims 9 and 10, Rajagopal and Liu do not explicitly discuss 5’ tail sequences on the primer which does not comprise the 5’ universal sequence.
Afonina teaches addition of 5’ tail sequences to either primer in real-time PCR (pg. 770, col. 3).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Afonina. One would have been motivated to do so in order to increase fluorescent signal (pg. 770, col 3).
Claims 12 and 13 are rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claims 1 and 11 above, and further in view of Wangh et al. (published March 18, 2004; Patent Application Publication US-20040053254-A1). It is noted that claim 11 was evidenced by Wangh et al.
Rajagopal and Liu teach the limitations of claims 1 and 11, as discussed above.
Regarding claims 12 and 13, Wangh teaches probes and extension products which form duplexes having melting temperatures higher than those for primers and the same sense extension products (par. 6).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Wangh. One would have been motivated to do so in order to design probes which do not interfere with primer extension (par. 6).
Claims 14-16 are rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claim 1 above, and further in view of Tsourkas et al. (published Oct 1, 2002; Tsourkas et al. Nucleic Acids Res. 2002 Oct 1;30(19):4208-15).
Rajagopal and Liu teach the limitations of claim 1, as discussed above.
Regarding claims 14-16, Liu teaches probes wherein the probes form a stem-loop and comprise 5’ to 3’: a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the ribonucleotide is in the loop sequence, and wherein the target-binding sequence comprises at least part of the loop sequence (Table 1; pg. 84, col 2: “Preparation of cMBs for HLA genotyping”)
Regarding claims 14-16, neither Liu nor Rajagopal explicitly teaches a 5’ universal sequence comprised in the loop and/or stem sequences of the probe.
Regarding claims 14 and 16, Tsourkas teaches conventional molecular beacons, wherein the probes form a stem-loop and comprise: a first stem sequence, a loop sequence, and a second stem sequence that is the reverse complement of the first stem sequence, wherein the target-binding sequence comprises at least part of or all of the loop sequence (Abstract; Table 1: “Conventional” probes).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Tsourkas. One would have been motivated to do so because probes having target-binding sequences within the loop may discriminate between targets with a relatively high specificity (Abstract).
Regarding claim 15, Tsourkas teaches ‘shared-stem molecular beacons’, wherein the target-binding sequence further comprises at least part of the second stem sequence (Abstract; Table 1: “Shared-stem” 19/4 and 19/7).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Tsourkas. One would have been motivated to do so because probes having one stem participating in target-binding form more stable duplexes than conventional probes (Abstract).
Claim 17 is rejected over the combination of Rajagopal et al (published June 24, 2021; Patent Application Publication US-20210189473-A1) and Liu et al (published March 1, 2010; Liu et al. Anal Biochem. 2010 Mar 1;398(1):83-92), as applied to claim 1 above, and further in view of Dobosy et al (published Feb 15, 2018; Patent Publication No. US 20180044716 A1).
Rajagopal and Liu teach the limitations of claim 1, as discussed above.
Regarding claim 17, Rajagopal does not recite that the RNase H2 enzyme originates from any specific organism, and Liu recites an RNase H2 enzyme from species not required by the claims.
Dobosy teaches mutants of RNase H2 enzymes from Pyrococcus furiosis, Pyrococcus horikoshii, Pyrococcus horikoshii, Thermococcus kodakarensis, and Thermococcus litoralis (par. 10).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Rajagopal and Liu with the teachings of Dobosy. One would have been motivated to do so in order to utilize RNase H2 enzymes which are capable of use in emulsion-based digital droplet PCR assays (par. 103).
Conclusion
No claims are allowed.
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/C.M.J./Examiner, Art Unit 1682
/AMANDA HANEY/Primary Examiner, Art Unit 1682
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