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 with traverse of Group I (claims 1-2, 5, 8-10, 13, 15, 18, 21-22, 27-29, and 44-47) in the reply filed on 12/9/2025 is acknowledged. The traversal is on the ground(s) that the groups share a special technical feature that is not taught by the prior art. This is not found persuasive because Applicant has amended claim 34 to now include the amplification kinetics limitation and the primer limitation of claim 1. These features are taught by Willey et al. (US 2015/0184240 A1) – see the rejection of claim 1 below.
The requirement is still deemed proper and is therefore made FINAL.
Claim 34 is therefore withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant has also added claims 48-50. Claims 1-2, 5, 8-10, 13, 15, 18, 21-22, 27-29, 44-50 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 4/11/2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner, except where noted.
The information disclosure statement filed 4/11/2024 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. A copy of the non-patent literature Rutledge (citation no. 12) has not been provided, and so this reference has not been considered.
Drawings
The drawings are objected to because in Figure 6, parts A, B, and C are listed, but in the description of this figure in the instant specification (page 77), these parts are not individually noted. The same issue is noted for Figure 12. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities: in the description of Figure 5 (page 77), parts A and B of the figure are listed, but in the drawings, Figure 5 does not have A and B labels. In the description of Figure 8 (page 77), parts A and B of the figure are listed, but in the drawings, Figure 8 does not have A and B labels. In the description of Figure 15 (pages 79-80), parts A, B, C, and D of the figure are listed, but in the drawings, Figure 15 does not have A, B, C, and D labels.
Appropriate correction is required.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Such hyperlinks appear on page 6, para. 6, page 7, para. 1, page 34, para. 4, page 35, para. 2, and page 94, para. 1 of the instant specification.
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, a distinction is made between amplification kinetics that are “not the same” as one another and amplification kinetics that are “not substantially similar” to one another. Thus, “the same” in the context of the claim will be interpreted as the amplification kinetics of two sequences being the exact same. 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 Objections
Claim 1 is objected to because of the following informalities: in line 1 of (a), “comprising the at least a first and at least a second target” should read “comprising a first and a second target,” as the phrasing “at least a/the” is not used to refer to the targets throughout the rest of the claim set. Additionally, the claim inherently does not preclude the use of additional targets, as the claim comprises the listed components. In line 2 of (a), “polynucleotides” should read “polynucleotide.” In line 2 of (c), “a first target polynucleotide” should read “the first target polynucleotide.” Finally, the word “and” should appear between the final two wherein clauses. Appropriate correction is required.
Claim 2 is objected to because of the following informality: in line 1, “wherein the method comprises” should read “wherein the method further comprises.” Appropriate correction is required.
Claim 5 is objected to because of the following informalities: in line 2 of (i) and line 2 of (ii), “at least first tuned competitor” and “at least second tuned competitor” should both have the “at least,” phrase removed, as this is not how the competitors are referred to elsewhere throughout the claim set. Additionally, in what appears to be line 2 of (ii), the phrase beginning with “is selected” is recommended to begin on a new line, as this limitation applies to both (i) and (ii). “Is selected” should also be amended to “are selected.” Finally, in line 2 of the “is selected” phrase, the “first target amplification product” and “second target amplification product” should both have the word “amplification” removed, as this is not how the products are referred to elsewhere in the claim set. Appropriate correction is required.
Claim 8 is objected to because of the following informalities: in the final phrase, “portion to be amplified” in line 1 and “to be amplified” in line 2 should be removed for clarity, as this language is not used in claim 1 to refer to these sequences. Appropriate correction is required.
Claim 9 is objected to because of the following informalities: the word “amplification” should be removed in “the first target amplification product” and “the second target amplification product,” as this language is not used in claim 1 to refer to these sequences. Appropriate correction is required.
Claim 10 is objected to because of the following informality: in line 1, “the method comprises” should read “the method further comprises.” Appropriate correction is required.
Claim 29 is objected to because of the following informalities: in line 3, a comma should appear after the word cancer. Additionally, as the phrase “human tuberculosis” is initially used in the claim, “human tuberculosis” should be used wherever only “tuberculosis” appears in the claim. In the second phrase (beginning with “the predictive relationship”), “the white blood cells” should simply read “white blood cells.” Finally, in the final line of the claim “patients” should read “subjects,” as this is the word used previously in the claim set. Appropriate correction is required.
Claim 44 is objected to because of the following informality: in line 2 of (a), “the target” should read ”the first target polynucleotide” to match the format used earlier in the claim. Appropriate correction is required.
Claim 45 is objected to because of the following informality: the phrase “polymerase chain reaction (PCR)” should simply read “PCR” to match the format used earlier in the claim set. 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.
Claims 5, 10, 13, 15, 18, 21, 28-29, 44, and 49-50 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 5 is rejected because the phrase “the predictive relationship of the target to one or more states” is unclear. “A predictive relationship” does not occur earlier in this claim or in claim 1, from which this claim depends. Thus, this phrase lacks sufficient antecedent basis. Additionally, “the target” is unclear because two target polynucleotides are utilized in the method of the claim, and so it is unknown which one is to be used as “the target,” or if either target polynucleotide may be used.
Claim 10 is rejected because the optional portion is unclear. Specifically, the optional portion includes (a), (b), and/or (c). In (a), the probe with the first label is capable of hybridizing to the first target product. In (b), it states that optionally, neither probe is capable of hybridizing to the first target product. It is thus unclear how a singular probe (i.e. the probe with the first label) would be both capable and incapable of hybridizing to the first target product.
Claims 13, 15, 18, and 21 are rejected based on their dependence on rejected claim 10.
Claim 18 is rejected because in lines 3-4, “the corresponding probe” and “the tuned competitor polynucleotide product” both lack sufficient antecedent basis. “A corresponding probe” is not discussed earlier in the claim or in claims 1 or 10, from which this claim depends. It will be interpreted as though “the corresponding probe” is referring to a second probe within the same probe group. “A tuned competitor polynucleotide product” is also not recited earlier in the claim or in claims 1 or 10, and in particular it is unclear whether this is referring to the first and/or second tuned competitor polynucleotide product. It will be interpreted that if the first or second target product is associated with a probe with a first label, the first or second tuned competitor product will be associated with the second label, respectively. It is noted that these terms also appear in the second scenario of claim 18, where similar indefiniteness issues surround the specific probe and tuned competitor product to be used. The same interpretation applied to the first scenario will also apply here.
Claim 28 is rejected because “the first label” and “the second label” both lack sufficient antecedent basis, as no labels of any kind are recited in claims 1 or 27, from which this claim depends. These labels will be interpreted to be any label used in conjunction with or on any of the sequences recited in claim 1.
Claim 29 is rejected due to its dependence on rejected claim 28.
Claim 29 is also rejected because in the second phase, “the predictive relationship, decision surface, or differential target oligonucleotide pattern” lacks sufficient antecedent basis, as these terms are not used earlier in the claim or in any of the claims from which this claim depends, and it is also unclear what these terms are referring to in relation to the components of the method of claim 1. The claim will be interpreted as though these values are derived from a white blood cell sample from a subject, but as to whether they refer to particular target polynucleotides or competitor polynucleotides, it is unknown. Prior art which thus reads on this limitation in light of any of the polynucleotides of claim 1 will be considered to meet this limitation.
In the final phrase of claim 29, differential regulation of three genes is recited, but these genes are not described in the claims previously, and so it is also unclear how they relate to the components of instant claim 1. In line 2 of this phrase, it is also unclear why “other disease” is in quotations marks, as it is unknown if this refers to any other disease, specific other diseases, or some other category, or if this is meant to impart a particular unknown limitation into the claim. Finally, in line 3 of this phrase, “the gene expression signature” lacks sufficient antecedent basis, and it is recommended that this phrase read “the gene regulation signature” as is used earlier in the claim (if these terms are intended to refer to the same concept/limitation).
Claim 44 is rejected because the claim recites options (a), (b), (c), and (d), where one or more of the options may be chosen (as evidenced by the use of and/or between options (c) and (d)). However, options (b) and (c) recite that the second primer is capable of hybridizing to the first and/or second tuned competitor polynucleotides, but (d)(ii) states that the second primer is not capable of hybridizing to either tuned competitor polynucleotide. It is thus unclear how a singular primer would be both capable and incapable of hybridizing to the first target product.
Claim 49 is rejected because the phrase “the same probe labeled with a first label” is unclear. Firstly, it is unclear if the “first label” on each probe must be the same for all probes, or if different first labels may be used for each probe, where each probe with the same sequence must simply contain at least one label. Put another way, it is unclear if the term “first label” refers to a singular label or a group of labels. The term “first label” seems to be used throughout the claims to refer to either option - for example, in claim 10, option (c), the “first label” is a particular singular type of fluorophore, but in claim 50, it appears the first label can also differ between probes (see the indefiniteness rejection of claim 50 below). Thus, prior art that reads on either interpretation of the term will be considered to meet this claim limitation. Secondly, the phrase “the same probe” in line 2 lacks sufficient antecedent basis, and “a probe” or “a same probe” is not recited earlier in the claim or in claims 1 and 48, from which this claim depends. It is recommended to amend the claim to first require that each competitor product is detected using a probe, and then require that the sequence of each probe is identical.
Claim 50 is rejected because the phrase “a different probe labelled with a first label” is unclear. Specifically, it is unclear if the “first label” on each probe must be the same for all probes, or if different first labels may be used for each probe, where each different probe must simply contain at least one label. Put another way, it is unclear if the term “first label” refers to a singular label or a group of labels. It will be interpreted as though the first label may be either the same label for all of the probes or a group of labels, as later in the claim it is optionally required that each label be the “same label,” indicating that this is not a requirement for the initial “a different probe labelled with a first label” language.
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 29 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 29 is directed to method of diagnosis or prognosis of a disease or condition, wherein in the optional claim limitations, the regulation of three genes (GBP6, ARG1, and TMCC1) is related to a probability of a subject having tuberculosis. The natural law recited is the relationship between the expression of these genes 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)(2). 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) and Levin et al. (US 2019/0323065 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). As the competitor template and the target nucleic acid differ by such a significant length, the amplification of each sequence would naturally slightly differ, and thus would not be the exact same. Therefore, this meets the amplification kinetics limitations of claim 1, as stated in accordance with the “Claim Interpretation” section above. 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.
Levin teaches methods of detecting active TB in a subject derived sample, where expression of GBP6, TMCC1, and ARG1 is used for said detection (paras. 14-16). These genes may also be detected via multiple PCR (paras. 44-45). The tuberculosis may be present with a cancer co-morbidity (para. 80). Blood samples can be utilized (paras. 56, 90, and 102, for example).
Thus, claim 29 is directed to a judicial exception without significantly more.
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, 27-29, and 44-48 are rejected under 35 U.S.C. 103 as being unpatentable over 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 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).
Willey is also silent to whether the competitive template of Example II would have different amplification kinetics to the target nucleic acid. However, as the competitor template and the target nucleic acid differ by such a significant length, the amplification of each sequence would naturally slightly differ, and thus would not be the exact same. Therefore, this meets the amplification kinetics limitations of claim 1, as stated in accordance with the “Claim Interpretation” section above.
Thus, claim 1 is prima facie obvious over Willey.
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).
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. 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.
Regarding claims 8 and 47, as the first competitor template 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.
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 cited above in the rejection of claim 1.
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.
Regarding claims 27 and 29, 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). 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 cited above in the rejection of claim 1. As detection with labels 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.
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.
Claims 10, 13, 15, 18, 21, and 50 are 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).
Regarding claims 10, 13, 15, 18, 21, and 50, Willey teaches the methods of claims 1-2, 5, 8-9, 22, 27-29, 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. 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, 15, and 50 are prima facie obvious over Willey 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 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, 15, and 50, 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 18 is prima facie obvious over Willey 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 Spier et al. (WO 2014/110528 A1).
Regarding claim 49, Willey teaches the methods of claims 1-2, 5, 8-9, 22, 27-29, 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. 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 Spier.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681