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 .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claim 34 remains withdrawn. Claim 29 has been canceled. Claims 1-2, 5, 8-10, 13, 15, 18, 21-22, 27-28, and 44-50 are pending and are considered on the merits.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 6/15/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
Drawing Objections
The drawings were objected to because several portions of the drawings were not individually noted in the specification. In light of Applicant’s amendments to the drawings submitted 7/21/2026, these objections have been withdrawn.
Specification Objections
The specification was objected to due to figure descriptions that did not match the provided drawings, and the use of hyperlinks. In In light of Applicant’s amendments to the drawings submitted 7/21/2026 and the amendments to the specification submitted 6/11/2026, these objections have been withdrawn.
Claim Objections
Claims 1-2, 5, 8-10, 29, and 44-45 were objected to for minor informalities. The objections for claims 2, 5, 8-10, and 44-45 have been withdrawn. The objections for claim 1 have been maintained-in-part, as part of the objection has not been amended by Applicant and is repeated below. Claim 29 has been canceled, and so this objection has been rendered moot. See also new grounds of objection below.
35 USC 112(b) Rejections
Claims 5, 10, 13, 15, 18, 21, 28-29, 44, and 49-50 were rejected for various indefiniteness issues. In light of the amendments to the claims submitted 6/11/2026, these rejections have been withdrawn for all currently pending claims. Claim 29 has been canceled, and so this rejection has been rendered moot.
35 USC 101 Rejections
Claim 29 was rejected for reciting a judicial exception without significantly more. Claim 29 has been canceled, and so this rejection has been rendered moot.
35 USC 103 Rejections
Claims 1-2, 5, 8-9, 22, 27-29, and 44-48 were rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1).
Claims 10, 13, 15, 18, 21, and 50 were rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1) in view of Thermo Fisher Scientific (“Essentials of Real-Time PCR,” 2020).
Claim 49 was rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1) in view of Spier et al. (WO 2014/110528 A1).
Applicant’s arguments and amendments have been thorough reviewed and considered. In light of the amendments to the claims submitted 6/11/2026, these rejections have been withdrawn for all currently pending claims, but see “Response to Applicant’s Arguments” and new grounds of rejection below. Claim 29 has been canceled, and so this rejection has been rendered moot.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that the amendment to claim 1, which now requires that the amplification kinetics of the first competitor and the first target not be “substantially similar” to one another, allegedly excludes the teachings of Willey (Remarks, page 20).
The Examiner agrees that the amendments to the instant claims change the scope in a manner that Willey does not address on its own. This is the basis for the new grounds of rejection below.
However, in interpreting the phrase “not substantially similar” as presented in instant claim 1, it is noted that the instant specification does not provide a specific definition for this term. Therefore, this term will be given its broadest reasonable interpretation. See “Claim Interpretation” below.
It is also noted that “amplification kinetics” is not defined in the instant specification. In considering this term in light of its broadest reasonable interpretation, the scope of the term is not precisely equal to that of “amplification efficiency.” As shown in Liu et al. (Analytical Biochemistry, 2002), amplification kinetics in PCR can refer to the general shape of the PCR curve, while amplification efficiency refers to a measure of general product yield as evaluated by a comparison of fluorescence measurements and cycle thresholds. Generally, amplification efficiencies are gleaned from the slope of the exponential phase of amplification and different fluorescent maxima (see Figure 1 and pages 53-54). Thus, it is possible for amplification efficiencies between two targets to be the same or similar, even when the overall kinetics of amplification differ.
Applicant argues that using Willey to arrive at the claimed invention would allegedly render the reference inoperable for its intended purpose, and though Willey teaches a target and competitor that are different lengths, the reference does not allegedly provide evidence to support its use in inherently reading on the amplification kinetic requirements of the instant claims. Applicant points to particular teachings of Willey to support this (Remarks, pages 22-23).
As noted above, the Examiner agrees that Willey alone does not provide teachings that read on the entirety of newly amended instant claim 1, particularly with regard to amplification kinetics. However, in regards to the teachings presented in the Non-Final Rejection, the Examiner utilized only teachings from Willey, with prima facie obvious rationale provided to arrive at a multiplex solution including the target and competitor of Example II and paras. 411-413, as multiplex reactions are taught elsewhere in the reference. It is unclear how only teachings utilized in the reference could render said reference inoperable for its intended purpose.
Applicant points to para. 80 of Willey which discusses amplification efficiencies of potential target and competitive templates to support their arguments. As noted above, amplification kinetics and amplification efficiency do not precisely occupy the same scope, though they are related terms. Furthermore, the teachings of para. 80 of Willey state that a “competitive template” generally refers to “a nucleic acid that competes with a target nucleic acid during an amplification reaction.” The amplification efficiency between the target and competitor is not necessarily required to be the same or substantially similar, as this is noted to be true in particular embodiments, and not for the invention as a whole. Additionally, the paragraph states, “a competitive template for a given nucleic acid can be amplified using one or more of the same primers as that of the given nucleic acid and/or amplifies with the same or substantially the same efficiency as the given nucleic acid. In preferred embodiment a competitive template for a given nucleic acid is amplified using the same primers, shares sequence homology, and/or amplifies with the same or substantially similar efficiency as the given nucleic acid,” (emphasis added). This indicates that the target and competitor, if amplified by the same primers and/or if sharing sequence homology, need not also have the same amplification efficiency, according to Willey. In the teachings used in the Non-Final Rejection, the target and competitor are amplified by the same primer pair, and so by the teachings of para. 80, need not necessarily have the same or similar amplification efficiency.
On pages 23-24, Applicant cites work by the inventors of the instant application (Goertz et al., bioRxiv, 2023; copy provided by Applicant) that demonstrates that amplification kinetics are based on a plurality of factors, and that length alone is not necessarily sufficient to demonstrate differences in kinetics. It is noted that Applicant does not point to particular citations in this reference.
Goertz teaches the use of competitive amplification networks (CANs; Abstract). In Figure 1B, a comparison of the natural transcript and the synthetic design is done. It is noted that while these natural and synthetic sequences are encompassed by the first target and competitor of instant claim 1, they do not read on the entirety of the scope of the claim, as the claim does not require fluorescent sequences to be inserted into the larger sequences. The caption to this figure reads, “At a fixed concentration of the synthetic amplicon, PCR amplification of different transcript concentrations leads to modulation of the steady state intensity of both probe fluorophores.” These transcript concentration differences produce the differences in amplification kinetics shown in the figure. Figure 2 shows that length and GC content, both alone and in combination, affect amplification rate (a term that is noted in the figure caption to be the steepness of the amplification curve, and so is related to amplification efficiency and kinetics). Figure S1 shows a similar pattern regarding length and GC content. Relating these figures to the teachings of Willey, and examining length alone in Goertz, while there are certainly length ranges in which there is little change in amplification efficiency, this rarely appears to be the case when the length change is 100bp. In Figure S1C for instance, the lines which show little change between 100 and 300bp appear to have no data points associated with them, or only a single data point, while large amounts of data points do appear to be associated with larger amplification rate changes. Additionally, in Willey, the target and competitor are based on the same general nucleic acid sequence for a particular gene. As the competitor is shorter than the target due to a deletion, provided the sequence deleted is not entirely composed of A/T nucleotides, the GC content of both the target and competitor will naturally differ, thus addressing both factors discussed by Goertz.
On pages 24-25, Applicant argues that Willey teaches away from the claimed invention, as Willey allegedly teaches that the 100bp length different is functionally neutral.
Regarding teaching away, MPEP 2145 X (D) 1 states, “‘the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….’ In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004). See also UCB, Inc. v. Actavis Labs, UT, Inc., 65 F.4th 679, 692, 2023 USPQ2d 448 (Fed. Cir. 2023) (‘a reference does not teach away if it merely expresses a general preference for an alternative invention but does not criticize, discredit or otherwise discourage investigation into the invention claimed.’).” Applicant points to para. 80 of Willey to support this point, but this paragraph does not disparage the use of competitors with different amplification efficiencies than targets, and as noted above, even encompasses the use of such competitors in its scope.
Overall, Applicant’s arguments are not persuasive to overcome the use of Willey generally in the context of prior art rejections. As noted above, the amendments to the claims require new grounds of rejection that are provided below, but the portions of the rejection in the Non-Final Rejection that remain relevant have been reiterated.
Claim Objections
Claim 1 is objected to because of the following informality: in line 2 of (a), “polynucleotides” should read “polynucleotide.”
Claim 18 is objected to because of the following informalities: in each option for the claim, the word “corresponding” should be inserted before “tuned competitor product,” forming the phrase “the corresponding tuned competitor product.” Appropriate correction is required.
Claim 50 is objected to because of the following informality: in line 3, “second tune” should read “second tuned.”. Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 28 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more. The claim recites a natural law.
Claim 28 is directed to method of diagnosis or prognosis of a disease or condition, wherein in the optional claim limitations, the amounts of the first and second labels may be correlated with a target sequence and its associated competitor. The natural law recited is the relationship between the amount of target label (and therefore, the expression of the target) present and the presence of disease in a subject. This judicial exception is not integrated into a practical application because there is no required active treatment step or other step that integrates the judicial exception into a practical application. See MPEP 2106.04(d). The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because they do not amount to more than well-understood, routine, and conventional activity in view of Willey et al. (US 2015/0184240 A1).
Willey teaches methods for evaluating nucleic acids and assessing the amounts of target in a sample (Abstract). The general method involves the co-amplification of targets and competitive templates, where more than one target and template may be used (paras. 6-10). Multiplex reactions may be used (thus containing multiple targets and competitors in a single reaction vessel; paras. 92 and 132). Example II shows the design of a competitor template, where the template is double-stranded and based on the sequence of a target nucleic acid, but is about 100 bp shorter (see Figure 32 and paras. 411-413). After the design of the competitor is complete, the competitor and target may be amplified together. It is noted that with this design the same forward and reverse primers can be used for both the target and competitor, as explained in para. 426. The amplification products for the target and competitor can then be analyzed (see paras. 429-434). Willey teaches in para. 13 that their invention also encompasses a database that nucleic acid target values can be compared to, where the database can correspond to a disease state. The database can comprise numerical values (para. 15) that incorporate values from multiple competitive templates (para. 19), and can involve relationships between nucleic acid targets and their respective competitive templates. A general comparative method for identifying disease states utilizing two nucleic acids per sample is also detailed in paras. 267-269. Paras. 270-271 specifically note that this disease state can be cancer. Willey also teaches that nucleic acids of interest can be quantified and detected in a sample (paras. 67-68). Additionally, paras. 109 and 177 teach that amplification products may be labeled with a detectable moiety such as a fluorescent moiety, and specifically that nucleic acid templates and competitive templates can be detected and compared quantitatively in this way.
Thus, claim 28 is directed to a judicial exception without significantly more.
Claim Interpretation
As stated in the application as published (US 2023/0366016 A1), para. 64 discusses the phrase “tuned.” It states, “The amplification is a “competitive” amplification that involves the use of a competitor polynucleotide that has been “tuned” to have particular features that are described herein.” Since the tuning involves general “specific features,” where said features are not defined, and so can include any features of a polynucleotide (e.g. length, sequence, GC content, etc.), any polynucleotide which can perform the claimed functions of the tuned polynucleotides will be considered to encompass a “tuned competitor polynucleotide.”
In instant claim 1, amplification kinetics between the first competitor and first target must be “not substantially similar” to one another. As stated above in the “Response to Applicant’s Arguments” section, there is no definition provided for “substantially similar” in the instant specification.” Thus, within the content of the instant claims, it will be interpreted that amplification kinetics are “not substantially similar” to one another if they can be readily distinguished from one another in the context of an amplification curve. It is noted that Applicant describes exemplar methods of altering amplification kinetics via GC content and length differences (paras. 165-167 of the specification as published).
Regarding instant claim 15, the term “label” is not defined by the instant specification. In the application as published, para. 18 mentions “fluorophore labeled probes,” and paras. 178-179 describe probes that are each labeled with “one of only two particular labels.” Para. 182 shows examples of such probes, where each probe has a fluorophore at one end and a quencher at another. Para. 183 also describes the reading of particular labels. Paras. 184-190 continue to describe labels, and in particular fluorescent labels. Para. 191 then states, “It will be clear then from the above that reference to a fluorophore does not mean that a quencher may not also be present.” Para. 456 also distinguishes the fluorophore labels from the quencher (i.e. the quencher is not referred to as a label). Thus, the term “label” will be given the broadest reasonable interpretation in light of the specification of an element that can be used to determine the presence or amount of a particular component. As quenchers are used to suppress detectable fluorescent labelling, quenchers that are not fluorescent themselves will not be considered labels for the purposes of applying prior art.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 5, 8-9, 22, and 44-48 are rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1) in view of Pionzio et al. (Forensic Science International: Genetics, 2014) and Debode et al. (Biotechnol. Agron. Soc. Environ., 2017).
Willey teaches methods for evaluating nucleic acids and assessing the amounts of target in a sample (Abstract). The general method involves the co-amplification of targets and competitive templates, where more than one target and template may be used (paras. 6-10). Multiplex reactions may be used (thus containing multiple targets and competitors in a single reaction vessel; paras. 92 and 132). Example II shows the design of a competitor template, where the template is double-stranded and based on the sequence of a target nucleic acid, but is about 100 bp shorter (see Figure 32 and paras. 411-413). After the design of the competitor is complete, the competitor and target may be amplified together. It is noted that with this design the same forward and reverse primers can then be used on the competitive template, as explained in para. 426. The amplification products for the target and competitor can then be analyzed (see paras. 429-434).
Though Willey does not teach that the target and competitor of Example II may be used in a multiplex reaction, as the use of multiplex reactions generally is taught in Willey, it would be prima facie obvious to utilize a multiplex reaction with the general method of Example II so that additional targets in a sample may be amplified along with additional competitor templates. For instance, if a user was interested in amplifying multiple target genes to examine allele frequencies, particular mutations, etc., it would save time and resources by having all of the targets be examined in a single reaction vessel. There would be a reasonable expectation of success as Willey both teaches multiplex amplification within the methods of the reference (see also paras. 386, 389, and 482, for example), and also notes that multiplex amplification is well-known in the prior art (e.g. paras. 132, 134, and 329).
However, Willey is silent to whether the competitive template of Example II would have different amplification kinetics to the target nucleic acid.
The differences between the competitor and target sequences of Willey amount to differences in amplicon length. Pionzio teaches that for a similar GC content, amplification kinetics changes with amplicon length. In Figure 1, each color of line represents the same treatment, while the different columns represent different GC contents. Each row is a different amplicon length. Within a GC content group, amplicon length affects amplification kinetics in each scenario where amplification occurs (i. e., when fluorescence is measured and not 0). A similar trend is shown in Figure 2, where each row is a different amplicon length for a single GC content, where different colors represent different treatments, and amplicon length clearly impacts amplification kinetics in each scenario. In Debode, similar results are shown. Figure 3 shows the creation of amplicons of different lengths, and Figure 4 shows amplification curves for the different amplicon lengths, where differences in kinetics can be seen for each length.
Thus, both Pionzio and Debode provide evidence that differences in amplicon length lead to differences in amplification kinetics. In both references, amplicon lengths that differed by 100bp were specifically shown to differ in their kinetics (e.g. Pionzio Figure 2 caption and Debode Figure 4). As these kinetic differences can be easily distinguished from one another on an amplification curve, they are considered to meet the “not substantially similar” requirement of instant claim 1, as described in the “Claim Interpretation” section above
Thus, claim 1 is prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claims 2 and 44-45, as noted above, in the amplification of Example II of Willey, primer pairs are used, which would involve the use of a second primer (para. 426). This primer pair would therefore have a forward and reverse primer capable of binding to the nucleic acid target and the competitive template in order to amplify them via PCR (see also the primer binding sites in Figure 32).
Thus, claims 2 and 44-45 are prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claim 5, Willey teaches the use of reference nucleic acids (e.g. paras. 6-9), and specifically teaches the use of multiple reference nucleic acid targets to be used in conjunction with multiple competitor templates for the reference nucleic acids (para. 8). The references chosen may be based on particular biological states (para. 89). The amount of amplified product of a target nucleic acid produced can be compared to that of a reference nucleic acid in ratio form, where the ratio of the amplified product of a nucleic acid to the amount of amplified product of its competitive template is compared to same ratio for the reference nucleic acid and its competitive template (paras. 93-96). Para. 97 notes that the relationship of these ratios can remain substantially constant beyond the exponential phase of amplification of the nucleic acid. Additional target nucleic acids can similarly be compared to the initial target (para. 98). These values may all be used for clinical diagnostic testing (para. 102). The competitive templates for both a target nucleic acid and a reference can be provided at known concentrations (para. 119), and therefore would be at known concentrations relative to one another (e.g. para. 19). This allows the target nucleic acid to be assessed relative to other nucleic acids being measured (para. 123). The state measured by Willey can be a disease state, where normal tissue can be used as a comparison (para. 268). The biological states can specifically be determined utilizing two samples and two nucleic acids within each sample (paras. 267-268).
Given these teachings, the ordinary artisan would be capable of arriving at the method of instant claim 5 in the method of Willey in view of Pionzio and Debode. Specifically, Willey teaches determining the amount of amplification product for given targets in a reaction, and particularly teaches doing so for a normal sample (rather than a diseased sample). Willey also teaches the use of reference nucleic acids, as well as keeping amplification ratios constant between particular targets/competitors in a sample. Thus, when trying to determine if a particular sample is a normal sample (i.e. when performing a diagnostic test), it would be prima facie obvious to keep sequences and concentrations of the targets and competitors the same as they were in the known normal sample, and to keep the amplification reaction conditions the same, so that amplification rates can be the same (in the case of no disease in the sample to be diagnosed) and a disease state determination can be made as accurately as possible. If this is not done, it could lead to false positive or false negative results for a disease, which can significantly impact patient care. As Willey recites determining amplification product amounts and concentrations, and teaches many mathematical computations throughout their disclosure (e.g. paras. 268, 277-278, and 289, as well as Figures 7 and 9), there would be a reasonable expectation of success.
Thus, claim 5 is prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claims 8 and 47, as the first competitor template of Willey described above is 100 bp shorter than the first target (see Figure 32), and each sequence is flanked by primer binding sequences, the first competitor amplification products would also be about 100 bp shorter than the first target amplification products.
Thus, claims 8 and 47 are prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claims 9 and 48, Willey teaches that nucleic acids of interest can be quantified and detected in a sample (paras. 67-68). Additionally, paras. 109 and 177 teach that amplification products may be labeled with a detectable moiety such as a fluorescent moiety, and specifically that nucleic acid templates and competitive templates can be detected and compared quantitatively in this way. In Example II of Willey, it is also stated that primers can be labeled with fluorescence (para. 426). Thus, as Willey generally teaches the labeling of the nucleic acid targets and competitor templates with detectable moieties for quantification, and teaches that the sequences in Example II may be detectably labeled, this would render it prima facie obvious to detect each of the nucleic acid targets and competitor templates in the method of Willey in view of Pionzio and Debode cited above in the rejection of claim 1.
Thus, claims 9 and 48 are prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claim 22, in para. 132 of Willey, regarding multiplex reactions, it states, “A nucleic acid and its competitive template may be co-amplified (and/or further co-amplified) in the same or different vessels as one or more other nucleic acid and corresponding competitive template,” (emphasis added). As an additional one or more nucleic acids/competitors may be used, this would involve multiplexing with a total of two or more nucleic acids/competitors, thus overlapping in scope with the multiplexing of the instant claim.
Thus, claim 22 is prima facie obvious over Willey in view of Pionzio and Debode.
Regarding claim 46, as noted above in the rejection of claims 2 and 44, Willey teaches a single primer pair that is capable of amplifying both a first target nucleic acid and its competitive template. However, the competitive template is not required to be amplified by the same primer pair as the target nucleic acid in all embodiments of Willey. Para. 80 specifically says that only one of the same primers used for the target nucleic acid can be used to amplify competitive templates (see also paras. 10-11). Willey also teaches that competitive templates can include long insertions or mutations compared to target nucleic acid sequences (para. 83). Thus, the target nucleic acid and the competitive template may only have a single primer in common. Such a scenario may occur when a further primer hybridizes to a non-identical portion of the target and competitive template (e.g. a portion of the competitive template that contains an insertion relative to a target), which would render a specific forward or reverse primer for both the target and competitive template. As “second primer” can simply be the second primer used in amplifying the first competitor polynucleotide in claim 44, the “second primer” here would be the primer specific to the unique competitive template sequence, and would thus not be capable of hybridizing to the target nucleic acid. The ordinary artisan may choose to design primers in this fashion to evaluate amplification efficiency for target sequences that contain deletions – for example, para. 296 describes a disease that results in large gene deletions, and notes that primers can be used to span this deleted region. Thus, in such an example, the target nucleic acid can be the disease gene, and the competitive template could be the normal gene that contains insertions relative to the disease gene. By designing primers specific to the portion of the sequenced affected by the deletion, it can be deduced with further certainty how much target nucleic acid is produced relative to a competitor, which may have clinical implications.
Thus, claim 46 is prima facie obvious over Willey in view of Pionzio and Debode.
Claims 10, 13, 15, 18, 21, 27-28, and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1), in view of Pionzio et al. (Forensic Science International: Genetics, 2014) and Debode et al. (Biotechnol. Agron. Soc. Environ., 2017), and further in view of Thermo Fisher Scientific (“Essentials of Real-Time PCR,” 2020).
Regarding claims 10, 13, 15, 18, 21, 27-28, and 50, Willey in view of Pionzio and Debode teaches the methods of claims 1-2, 5, 8-9, 22, and 44-48, as described above. Willey also teaches that real-time PCR methods may be utilized in their invention, which make use of fluorescent probes (paras. 136 and 247). However, the reference does not teach the specifics of real-time PCR with their methods.
Thermo Fisher Scientific teaches the basics concerning real-time PCR with TaqMan probes. The reference teaches that these probes are each developed for a specific PCR product, and contain a fluorophore and quencher. The probes produce fluorescence as amplification proceeds by releasing the fluorophore from the proximity of the quencher (see “Step Process” and the figure below it). Thermo Fisher Scientific teaches the advantages of this method in that there is no post-PCR processing required and that the probe specifically hybridizes to a target, which would reduce non-specific signal. The reference also states that when analyzing multiple sequences simultaneously, probes can be labeled with different, distinguishable reporter dyes, and that for distinguishing different sequences, the creation of different probes would be required (“Advantages of TaqMan Chemistry” and “Disadvantage of TaqMan Chemistry”).
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 Thermo Fisher Scientific to perform the recited real-time PCR in Willey in view of Pionzio and Debode. Willey, as noted above, already teaches the use of real-time PCR, and Thermo Fisher Scientific simply provides additional guidance about how this method is typically performed. In Willey, the real-time PCR would be performed on at least two target nucleic acid/competitive templates simultaneously in the multiplex reaction described above, and the reference already teaches comparing the fluorescence of different amplified products (e.g. para. 177). Thus, the ordinary artisan would be capable of designing specific probes with unique labels for each target and competitive template, in order to ensure that each amplified sequence could be distinctly measured and analyzed in the amplified product. If probes or labels were not able to distinguish between the amplified products of different targets and/or competitive templates, then the different target genes could not be compared to one another, which may limit utility for diagnostic and other clinical applications. As the targets and their respective competitive templates are not identical sequences (i.e. the competitive templates are shorter), there would be a reasonable expectation of success in designing distinct probe for each of the sequences in the multiplex reaction.
It is noted that the distinct probes with unique labels can be considered two different probe groups as stated in instant claim 10 – where each probe group is specific to a target nucleic acid/competitive template pair. There would thus be two probe groups, as stated in (b) of instant claim 13. In Thermo Fisher Scientific, it is shown in the TaqMan process figure that only the single fluorophore label is used for each probe, and the quencher does not produce any fluorescence. Therefore, each probe is considered to have a single label, as stated in the “Claim Interpretation” section above, and so reads on claim 15.
Thus, claims 10, 13, and 15 are prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific.
Further regarding claim 18, as stated above in the rejection of claim 10, the probe groups can be considered to be the probes for each target nucleic acid/competitor pair, where two labels are used, one for the target and the other for the competitor. Willey also generally teaches that the target nucleic acids used can be predicative for a particular biological state, such as a disease state. A general comparative method for identifying disease states utilizing two nucleic acids per sample is also detailed in paras. 267-269. In such an example where the targets are chosen to predict a disease state, the amount of each target produced during amplification (that would be detected via the real-time PCR probes) would have either a positive or negative predictive relationship for said disease state, depending on if the targets were upregulated or downregulated for a particular disease. As Willey teaches that a normal sample can be used for comparison with a sample suspected of disease, and generally teaches that their method can be used for diagnostics (see paras. 102, 253, 263, 265, and 271, for example), such an application of their method would be prima facie obvious and within the capability of the ordinary artisan.
Thus, claim 18 is prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific.
Further regarding claim 21, Willey teaches that amplification products may also be detected via the use of gel electrophoresis. In such methods, the relative fraction of each sample for at least two targets was measured (see Figures 34 and 35, as well as paras. 500 and 504). Specifically, total optical value of both bands in a sample was then used to determine the relative values for each band (para. 504). This was then used to find the ratios of the targets to one another in the sample.
In considering this methodology in light of the real-time PCR methods described above in the rejection of Willey in view of Thermo Fisher Scientific for claims 10, 13, and 15, it would be prima facie obvious to incorporate relative fluorescent values into analysis of real-time PCR results to more usefully apply said results. The relative fluorescence of the two targets within the total reaction mixture could be measured, as well as the relative fluorescence of the two competitor templates to one another and the relative fluorescence of the target/competitor ratios to one another. These values would be useful in noting particular differences between the targets within different types of samples (such as those with early versus late stage disease). As the fluorescence values for each target and competitive template are already measured during the real-time PCR, this would amount to a simple additional mathematical analysis that one of ordinary skill in the art would be capable of.
Thus, claim 21 is prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific.
Regarding claim 27-28, Willey teaches in para. 13 that their invention also encompasses a database that nucleic acid target values can be compared to, where the database can correspond to a disease state. The database can comprise numerical values (para. 15) that incorporate values from multiple competitive templates (para. 19), and can involve relationships between nucleic acid targets and their respective competitive templates. A general comparative method for identifying disease states utilizing two nucleic acids per sample is also detailed in paras. 267-269. Paras. 270-271 specifically notes that this disease state can be cancer.
Regarding claim 28 specifically, Willey teaches that nucleic acids of interest can be quantified and detected in a sample (paras. 67-68). Para. 108 notes that probes can be used to quantify products, and para. 136 notes that fluorescent probes typically used in real-time RT-PCR may be employed (para. 136). As noted above, Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific renders prima facie obvious the use of real-time PCR with target specific fluorescently labeled probes. As this real-time PCR detection would be prima facie obvious, it would also be obvious to incorporate this detection into the disease state determination of Willey described above, so that the detection can have clinical and practical relevance, and may be able to aid in diagnosing particular conditions, which can affect patient treatment plans and outcomes.
Thus, claim 27-28 are prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific.
Regarding claim 50, the ordinary artisan would recognize that while individual fluorophores can be used to detect particular sequences in real-time PCR, cumulative detection, where all fluorophore detection is taken into account at once, could also be done. Such cumulative presence detection would be useful for quick processing of multiple target nucleic acids/competitor templates, where it is simply important to determine the total amount of amplified product. Such a scenario may be desired when comparing samples to one another, such as is described by Willey (para. 267). For example, if a sample for a normal subject has a particular cumulative presence, then cumulative presence could be quickly determined for sample suspected of having a disease, and based on whether the results were different, this general metric could then inform if additional diagnostic testing is necessary. In such a case, the ordinary artisan, using ordinary skill, creativity, and knowledge in the art, would recognize that the sequence specific probes taught by TaqMan could all be used with the same fluorophore. This would eliminate the need to add together each fluorophore intensity individually, and would also cut down on probe design complexity, as potential interference between fluorophore wavelengths would no longer be relevant. As this would involve using the same probe sequences taught in Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific above, there would be a reasonable expectation of success.
Thus, claim 50 is prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Thermo Fisher Scientific.
Claim 49 is rejected under 35 U.S.C. 103 as being unpatentable over Willey et al. (US 2015/0184240 A1), in view of Pionzio et al. (Forensic Science International: Genetics, 2014) and Debode et al. (Biotechnol. Agron. Soc. Environ., 2017), and further in view of Spier et al. (WO 2014/110528 A1).
Regarding claim 49, Willey in view of Pionzio and Debode teaches the methods of claims 1-2, 5, 8-9, 22, and 44-48, as described above. Willey also teaches that real-time PCR methods may be utilized in their invention, which make use of fluorescent probes (paras. 136 and 247). However, the reference does not teach the specifics of real-time PCR with their methods.
Spier teaches compositions for performing multiplex real-time PCR (Abstract). Target-specific primers are used in a multiplex fashion, where targets that are intended to be detected together comprise the same universal tags, which can be detected using labeled probes (para. 21). Figure 1 shows a basic outline of the method, where the primers incorporate the tags into each nucleic acid target (where the maximum number of targets is not specified; para. 32). The tagged amplicons can then be detected via TaqMan chemistry with labeled probes (option d in Figure 1). Para. 43 states that each universal probe can contain a different dye for each of the multiple targets, though the tag for each can be the same, allowing for cumulative presence detection. The same dye can also be used for multiple targets that have the same tag (para. 19).
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 Spier to incorporate universal tagged primers and probes into the real-time PCR of Willey in view of Pionzio and Debode. Specifically, this would involve adding universal sequences to the real-time PCR primers of Willey, and then using TaqMan probes specific to that universal sequence. As Spier teaches that detection is done based on the number of tags, the labels on the TaqMan probes may be the same or different for each target, but both would result in cumulative presence detection. This would be useful for quick processing of multiple target nucleic acids/competitor templates, where it is simply important to determine the total amount of amplified product. Such a scenario may be desired when comparing samples to one another, such as is described by Willey (para. 267). For example, if a sample for a normal subject has a particular cumulative presence, then cumulative presence could be quickly determined for sample suspected of having a disease, and based on whether the results were different, this general metric could then inform if additional diagnostic testing is necessary. The use of tagged primers and universal probes would have a reasonable expectation of success as Spier already teaches them within the context of multiplex real-time PCR.
Thus, claim 49 is prima facie obvious over Willey, in view of Pionzio and Debode, and further in view of Spier.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/F.F.G./Examiner, Art Unit 1681
/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1681
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