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 .
Status of Claims
Applicant’s amendment filed 05/08/2023 is acknowledged. Claims 3-9, 11, and 13-17 have been amended. Claims 1-17 are pending in the instant application and the subject of this non-final office action.
Specification
The disclosure is objected to because of the following informalities:
The specification attempts to incorporate by reference mutations 6223 and 6210 from the COSMIC database. See the 112(b) rejection below. This incorporation by reference is not effective because this represents essential material, as these are claimed in claim 12, and the COSMIC database is not a US patent or US patent application. See 37 CFR 1.57 and MPEP 2172.01.
Under rule 1.57(g), Applicant must amend the specification to include the essential material incorporated by reference (e.g., specific sequences and/or specific coordinates with the corresponding genome assembly/transcript/etc. utilized by the database corresponding to each mutation).
Appropriate correction is required.
Applicant the attempt to incorporate the material of WO 2019/165469 A1 is also noted. See 112(a) rejection below. Applicant is similarly warned that essential material from this document must be amended into the specification.
Claim Objections
Claims 1 and 2 are objected to because of the following informalities:
Claims 1 and 2 appear to have extra space(s) located at “the first polynucleotide is present” (obtaining paragraph) and ”identifying the emission”, respectively.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claim 1, “responsive to the site of the mutation” is defined as “provides a detectable response or signal, or change in response or signal, that is dependent on the presence of the specific mutation” (para [0044]).
Regarding claim 2, it is noted that “a base of the probe sequence that is complementary to the second polynucleotide” does not preclude complementarity to the first polynucleotide.
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 1-17 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 claim 1, first, the claim recites “identifying an emission from the probe sequence with a signal to noise ratio exceeding 10 when at least one copy of the second polynucleotide is present in the sample”. The nexuses is unclear between the emission and the probe sequence. See MPEP 2172.01 and instant para [0010].
The step of identifying is only time-limited in so far as “when at least one copy of the second polynucleotide is present in the sample”. It is not clear from the current claims if the emission is intended to occur as a result of hybridization, e.g., from the reporter responsive to the mutation, or from some other undisclosed part of the probe sequence. The summary of the invention, however, states that “[l]ight emission from the probe sequence, is then measured, wherein the mission has a signal to noise ratio greater than 10 …” (para [0010]).
Second, it is unclear how to calculate a “signal to noise ratio” with “an emission”. While it may be understood that the emission is the “signal” of the “signal to noise ratio”, it is not clear what the “noise” is intended to be used. The artisan would generally understand that an emission would be detected from the probe and a signal to noise ratio could be calculated based on other emissions detected that form a noise “floor” (e.g., from background fluorescence from the system/unbound probe). With only one emission, it is unclear how a ratio may be calculated. Thus, the metes and bounds are unclear to one of ordinary skill in the art.
Third, the claim recites “at least partially suppressing … to generate an amplified first polynucleotide and an amplified second polynucleotide”. The claim is indefinite because the requisite degree to which amplification must be suppressed and maximum bounds of suppression is not clear. The term “at least partially” encompasses fully but the claim also recites generating both a first and second nucleotide through the amplification. Thus, the metes and bounds are not clear to one of ordinary skill in art.
Claims 2-17 are indefinite for depending from claim 1 and not rectifying the deficiencies.
Regarding claim 2, the claim recites “the linker”. There is insufficient antecedent basis for this limitation in the claim.
Regarding claim 12, the claim recites “the mutation comprises an EGRF L858R mutation, an Exon 19 deletion, a C6223 mutation, and a C#6210 deletion.”
First, it is not clear which mutation(s) are being claimed. The claim limitation states that the mutation comprises … an Exon 19 deletion, a C6223 mutation, and a C#6210 deletion. It is unclear if “and” was intended or “or” as, according to Table 1, C6223 and C#6210 are Exon 19 deletions. Further, no examples were identified of detecting multiple types of mutations located across or how a reporter would be responsive to separate mutations.
Second, the EGFR mutations “C6223” and “C#6210” appear to be a reference to the COSMIC database. See Table 1 and para [0010] of the instant specification. While inclusion of the COSMIC database mutations qualifies as an intent to incorporate specific mutations/sequences, these identifiers fail to uniquely identify the specific mutation/sequence intended to be encompassed by the claims because there external databases may change the mutation(s) to which identifiers refer in different versions and, as claimed material, such represents essential material”. which may only be incorporated by reference from a US patent or US patent application publication that does not itself incorporate such material by reference. See 37 CFR 1.57 and 2172.01.
Thus, the claim is indefinite because it fails to specifically point out the mutations/sequences that are encompassed by the claim.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 1-3, 6-7, 9, and 11-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
In analyzing the claims for compliance with the written description requirement of 35 U.S.C. 112, first paragraph, the written description guidelines note that with regard to genus/species situations, a “satisfactory disclosure of a “representative number'' depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features of the elements possessed by the members of the genus in view of the species disclosed.”
Regarding 1-3, 6-7, 9, and 11-17, claim 1 requires 1) that an amplification reaction uses a primer pair complementary to both the first and the second polynucleotides to generate amplified polynucleotide comprising both amplified polynucleotides with and without the mutation; 2) that the probe sequence comprise a polynucleotide complementary to at least a portion of the amplified polynucleotide comprising the mutant and the amplified polynucleotide comprising the wild type sequence; and that 3) the reporter of the probe “sequence” is “responsive to the mutation” when the probe “sequence” is hybridized to the amplified polynucleotide comprising the mutation.
The mutations of the instant disclosure are described broadly in the specification to include a deletion or a single nucleotide polymorphism, a transposition, a translocation, and/or an insertion (para [0010]), and defined in the claims 4-7 to comprise each of those. In particular, claim 6 recites that the “mutation” comprises a transposition and claim 7 recites that the “mutation” comprises a translocation.
Absent a definition or examples in the specification, a transposition “mutation” is interpreted to be a movement of a transposable element (Wells JN, Feschotte C. A Field Guide to Eukaryotic Transposable Elements. Annu Rev Genet. 2020 Nov 23;54:539-561. Epub 2020 Sep 21.: entire document, e.g., Keywords; Introduction, para 1), and a translocation “mutation” is interpreted to be a chromosomal translocation, i.e., movement of a chromosomal segment from one chromosome to another or to a different location on the same chromosome (Massive Bio. Translocation [Internet]. Massive Bio; 2025 [cited 2026 Jan 4]. Available from: https://massivebio.com/translocation-bio/).
The working examples present are directed to a SNP or small deletions. No examples were identified with representative species described or presented for transposable elements, translocations, or other “large” mutations.
The specification discloses two sets of primers in the working examples in Table 2 directed to the EGFR exon 19 deletion (SEQ ID NO: 1-2, aka 3-4) and to KRAS G12 (SEQ ID NO: 5-6, aka 7-8, 9-10, …, and 15-16. An in silico PCR was performed using the tool available on the UCSC Genome Browser for assembly hg38 (Located at: genome.ucsc.edu/cgi-bin/hgPcr?hgsid=3536498085_vJNPTcr1jZW5yMHVNV5wkREFAada) for each set of PCR primers. SEQ ID NO: 1-2 produced an amplicon at location chr7:55174741+55174843 with length 103bp. SEQ ID NO: 5-6 produced an amplicon at location chr12:25245230-25245395 with length 166bp.
Su (Su J, et al. Molecular characteristics and clinical outcomes of EGFR exon 19 indel subtypes to EGFR TKIs in NSCLC patients. Oncotarget. 2017 Nov 30;8(67):111246-111257: Table 1) teaches that the EGFR Exon 19 deletions probes of Table 19 target deletions of 12 and 15 bases, respectively. The largest probe in the examples of Table 4 is 27 nucleotides (SEQ ID NO: 25).
The specification also recites that the “SNP-Switch” of the instant ssPCR of the working examples is Compound 25 of WO 2019/165469 A1 (e.g., para [0010], [0050-66], Tables 4 and 5; see para [0045]). This document recites throughout that the probes of that invention are capable of detecting SNPs or small insertions or deletions 1-20 nucleotides in length (e.g., para [0004], [0014], and [0020], claim 13). There is no indication in the current disclosure that the probes/reporter have been modified in such a way as to change the responsive portion of the probe to expand the mutation. And while another compound is recited, there is no indication it was used in the working examples, which recite “ssPCR”.
In contrast to the SNP and small deletions described in the working examples, Wells teaches that transposable elements typically range in length from 100 to 10,000 base pairs, but are sometimes far larger (Introduction, para 1). Massive Bio teaches that translocations involve pieces of chromosomes, arms of chromosomes, and/or segments of chromosomes (Types of Chromosomal Translation), i.e., on the scale of megabases. Indeed, Kodama (Kodama, Y., et al. Estimation of minimal size of translocated chromosome segments detectable by fluorescence in situ hybridization. International Journal of Radiation Biology. 1997 Jan;71(1):35–9) teaches that minimal delectable sizes of translocations for atomic bomb survivors were ~11 Mb. (Abstract).
This large difference in the size of the “mutation” presents the first source of variability because it has not been made clear to the artisan how the Applicants intend for the artisan to probe amplified polynucleotides comprising 100 to 10,000 bp, and sometimes larger (transposition), let alone many megabase (translocation) “mutations” when the exemplary probes are less than or equal to 27 bp for amplicons <200 bp, particularly when the probes using the same technology as the instant invention have been previously described only to detect mutations up to 20 bp.
Further, the claims also require amplifying both species of the polynucleotides comprising and not comprising the mutation and identifying a required signal to noise ratio, which appears to have been intended to be directed to hybridization of the probe “sequence”.
The artisan would expect that maintaining such would require an expectable efficiency of the reaction for the mutated polynucleotide. ResearchGate (Why the size of Amplicons 80–200 bp are acceptable in real-time PCR? [Internet] ResearchGate; 2016 [cited 2026 Jan 5]. Available from: https://www.researchgate.net/post/Why_the_size_of_amplicons_80-200_bp_are_acceptable_in_real-time_PCR) teaches that the efficiency of PCR noticeably decrease outside of ~200 bp and that such decreases in efficiency may interfere with comparative analyses.
It follows that the artisan would expect a drop of in amplification efficiency for mutations that dramatically extend the length of the amplicon such as transposons and translocations. Indeed, it is not clear that it is even possible to design primers to produce amplicons wherein the primers may be complementary to both species for many case of at least translocations (e.g., where the breakpoint is an arm of a chromosome, for example, and no flanking sequence on one side could be considered “wild type”).
Additionally, claim 12 recites “the mutation comprises an EGRF L858R mutation, an Exon 19 deletion, a C6223 mutation, and a C#6210 deletion”. Su teaches that the C6223 and C6210 mutations involve alterations of the nucleic acids that encode amino acids 750 and 751, respectively (Table 2). Even such methods were directed at the RNA, with another 858 mutation, such a probe would need to be “responsive” to mutations spanning ~325 nt. As above, no such examples of multiple mutation or probes/amplicons directed at such large “mutations” were described in working examples.
Accordingly, even for the smaller transposable elements, given the decreased amplification efficiency observed at a difference of 100 bp between amplicons, according to Research Gate, and the potential for difference in signal-to-noise given such vastly different probe lengths that would be needed, the species provided are not representative of large “mutations” (e.g., transposition events, translocations, multiple SNPs/indels spanning large regions, etc.). Thus, the skilled artisan would not have reasonably concluded that the Applicant had possession of the full scope of the claimed invention of identifying both large and small mutations as claimed at the time of the effective filing date.
For this reason, the claims do not comply with the written description requirements of 112(a).
Claims 1-3, 6-7, 9, and 11-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for small mutations (SNPs and indels), does not reasonably provide enablement for large mutations (>=21 bp) or collections of mutations spanning such distances. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988). Wands states, on page 1404:
Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex part Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of these in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
Regarding claims 1-3, 6-7, 9, and 11-17, first, the claims are broad. Claim 1 requires 1) that an amplification reaction uses a primer pair complementary to both the first and the second polynucleotides to generate amplified polynucleotide comprising both amplified polynucleotides with and without the mutation; 2) that the probe sequence comprise a polynucleotide complementary to at least a portion of the amplified polynucleotide comprising the mutant and the amplified polynucleotide comprising the wild type sequence; and that 3) the reporter of the probe “sequence” is “responsive to the mutation” when the probe “sequence” is hybridized to the amplified polynucleotide comprising the mutation.
Otherwise, they may apply to any sample wherein the first wild type polynucleotide is present in 100-fold excess (or greater for claims 13-15), but of any sample type/source. The suppression may be accomplished by any means (aside from claim 17, which remains broad so long as a primer includes PNA, LNA, or XNA). The reporter of the probe may encompass a wide variety, so long as it is “responsive to the mutation”.
Further, the claims are directed not only to the described SNPs and small deletions, but also classes of “large” mutations defined in claims 6 and 7 and a group of mutations in claim 12.
Second, the invention is in the class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., Inc. v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
Third, the guidance is limited to SNPs and small deletions. No examples were identified with representative species described or presented for transposable elements, translocations, or groups of mutations in the same amplification (wherein the mutations may be considered “the mutation”).
The specification discloses two sets of primers in the working examples in Table 2 directed to the EGFR exon 19 deletion (SEQ ID NO: 1-2, aka 3-4) and to KRAS G12 (SEQ ID NO: 5-6, aka 7-8, 9-10, …, and 15-16. An in silico PCR was performed using the tool available on the UCSC Genome Browser for assembly hg38 (Located at: genome.ucsc.edu/cgi-bin/hgPcr?hgsid=3536498085_vJNPTcr1jZW5yMHVNV5wkREFAada) for each set of PCR primers. SEQ ID NO: 1-2 produced an amplicon at location chr7:55174741+55174843 with length 103bp. SEQ ID NO: 5-6 produced an amplicon at location chr12:25245230-25245395 with length 166bp.
Su (Su J, et al. Molecular characteristics and clinical outcomes of EGFR exon 19 indel subtypes to EGFR TKIs in NSCLC patients. Oncotarget. 2017 Nov 30;8(67):111246-111257: Table 1) teaches that the EGFR Exon 19 deletions probes of Table 19 target deletions of 12 and 15 bases, respectively. The largest probe in the examples of Table 4 is 27 nucleotides (SEQ ID NO: 25).
The specification also recites that the “SNP-Switch” of the instant ssPCR of the working examples is Compound 25 of WO 2019/165469 A1 (e.g., para [0010], [0050-66], Tables 4 and 5; see para [0045]). This document recites throughout that the probes of that invention are capable of detecting SNPs or small insertions or deletions 1-20 nucleotides in length (e.g., para [0004], [0014], and [0020], claim 13). There is no indication in the current disclosure that the probes/reporter have been modified in such a way as to change the responsive portion of the probe to expand the mutation. And while another compound is recited, there is no indication it was used in the working examples, which recite “ssPCR”.
In contrast to the SNP and small deletions described in the working examples, the art teaches that “transposition” mutations and translocation “mutations” are long. Wells teaches that transposable elements typically range in length from 100 to 10,000 base pairs, but are sometimes far larger (Introduction, para 1). Massive Bio teaches that translocations involve pieces of chromosomes, arms of chromosomes, and/or segments of chromosomes (Types of Chromosomal Translation), i.e., on the scale of megabases. Indeed, Kodama (Kodama, Y., et al. Estimation of minimal size of translocated chromosome segments detectable by fluorescence in situ hybridization. International Journal of Radiation Biology. 1997 Jan;71(1):35–9) teaches that minimal delectable sizes of translocations for atomic bomb survivors were ~11 Mb. (Abstract).
This large difference in the size of the “mutation” presents the first source of unpredictability because it has not been made clear to the artisan how the Applicants intend for the artisan to probe amplified polynucleotides comprising 100 to 10,000 bp, and sometimes larger (transposition), let alone many megabase (translocation) “mutations” when the exemplary probes are less than or equal to 27 bp for amplicons <200 bp. No direction was identified for how the artisan is intended to target such “large” mutations with the probes of the invention that are required to be complementary to at least a portion of both the first and second polynucleotide and “responsive to the mutation”. Indeed, the probes of Table 4 place the mutation in the center of the probe.
Similarly, claim 12 recites “the mutation comprises an EGRF L858R mutation, an Exon 19 deletion, a C6223 mutation, and a C#6210 deletion”. Su teaches that the C6223 and C6210 mutations involve alterations of the nucleic acids that encode amino acids 750 and 751, respectively (Table 2). Even such methods were directed at the RNA, with another 858 mutation, such a probe would need to be “responsive” to mutations spanning ~325 nt. No examples of multiple mutation or probes/amplicons directed at such large “mutations” identified.
Accordingly, there would be a high level of experimentation required to determine whether the probes of the disclosure would function and function at the level of the required claimed signal to noise ratio, which appears to be intended to be linked with the reporter and/or hybridization of the probe.
Further, the claims also require amplifying both species of the polynucleotides comprising and not comprising the mutation with a required signal to noise ratio, which appears to have been intended to be directed to hybridization of the probe “sequence”. No working examples were identified of amplicons greater than 166 bp.
The art teaches that amplifying a polynucleotide with a mutation that greatly increases the amplicon size would introduce a high level of unpredictability to the results because such interferes with the efficiency of the amplification. ResearchGate (Why the size of Amplicons 80–200 bp are acceptable in real-time PCR? [Internet] ResearchGate; 2016 [cited 2026 Jan 5]. Available from: https://www.researchgate.net/post/Why_the_size_of_amplicons_80-200_bp_are_acceptable_in_real-time_PCR) teaches that the efficiency of PCR noticeably decrease outside of ~200 bp and that such decreases in efficiency may interfere with comparative analyses. Thus, the artisan would expect that amplifying “large” mutations would decrease the efficiency of the reaction for the mutated polynucleotide relative to the smaller wild type amplicon, which would be expected to decrease some measure of signal-to-noise, introducing unpredictability. Further, it is not clear that it is even possible to design primers to produce amplicons wherein the primers may be complementary to both species for many case of at least translocations (e.g., where the breakpoint is an arm of a chromosome, for example, and no flanking sequence on one side could be considered “wild type”).
Accordingly, even for the smaller transposable elements, given the decreased amplification efficiency observed at a difference of 100 bp between amplicons, according to Research Gate, and the potential for difference in signal-to-noise given such vastly different probe lengths that would be needed, the species provided are not representative of the claimed transposition and translocation “mutations”. Thus, there would be a high level of corresponding experimentation to determine the bounds of efficiency that would enable the artisan to achieve the signal to noise ratio given particular size ranges of mutations (e.g., classes of transposons) across the broad scope of the samples, reporters, and suppression mechanisms claimed.
Taken together, there is a high degree of uncertainty and experimentation required due to the 1) breath of all possible combinations of sample types, probes/reporters, and suppression means; 2) general unpredictability of the art; 3) teachings of the art that a) the transposition and translocation mutations differ in scope from the SNPs and indels described and that b) amplification efficiency can drop readily in similar detection methods; and 4) limited guidance directed solely to SNPs and small deletion mutations with no identified guidance for larger scale mutations. Balanced only against the high skill level in the art, it is held that the level of experimentation to utilize the full scope of the invention claimed would be undue.
For this reason, the claims do not comply with the 112(a) enablement requirements.
It is further noted that the specification recites “SNP-Switch” from international patent application international patent application WO 2019/165469 A1, as cited above. The attempt to attempt to incorporate the material in para [0001] is recognized.
If applicant decides to amend particular features of SNP-Switch and/or Compound 25 of WO 2019/165469 A1, such an incorporation by reference would not be effective under 37 CFR 1.57 7 as it 1) is not a US patent or US patent application publication and 2) provides support for claimed features. All material required to support the claimed inventions must be amended into the specification. See 37 CFR 1.57(g).
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.
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.
Claim(s) 1-5, 8-15, and 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Princen (US 2014/0248612 A1; published 09/04/2014; as cited in the IDS dated 09/19/2024) in view of Hanna (WO 2019/165469 A1; published 08/29/2019).
Regarding claim 1, Prince teaches a method of identifying a mutation of a wild type polynucleotide (entire document, e.g., claims 1-16; para [0159-292], Examples 1-9), comprising:
obtaining a sample comprising a first polynucleotide comprising the wild type polynucleotide and a second polynucleotide comprising the mutation, and wherein the first polynucleotide is present in at least a 100-fold excess over the second polynucleotide (para [0251], wherein a sample must have been obtained in order to include it in the reaction mixture; para [0184]: fold excess; see also [para [0111-112]);
performing an amplification reaction on the sample while at least partially suppressing amplification of the first polynucleotide and not suppressing amplification of the second polynucleotide, using a primer pair complementary to both the first and second polynucleotides to generate an amplified first polynucleotide and an amplified second polynucleotide (para [0252-253]; [0256]; see para [0194] and [0213]);
contacting the amplified sample with a probe “sequence” comprising a polynucleotide complementary to at least a portion of the amplified first polynucleotide and the amplified second polynucleotide (para [0254]; see [0228-236], i.e., “locus-specific detector probes” and Fig. 27A, which illustrates a probe sequence complementary to a portion of both polynucleotides, wherein the artisan would understand that the detector probes would likewise share such complementarity to the targets); and
identifying an emission from the probe sequence with a signal to noise ratio exceeding 10 when at least one copy of the second polynucleotide is present in the sample (para [0257]; signal-to-noise: e.g., Fig. 1-10 wherein the “emissions” generated by the detector probes exceed the background “noise” by at least a factor of 10).
Princen teaches that the detector probes may be TaqMan probes, various molecular beacons, Scorpion probes, PNA light-light up probes, and probes comprising reporter dyes and a quencher(para [0109]).
Princen fails to explicitly teach a reporter that is responsive to the mutation when the probe sequence is hybridized to the amplified second polynucleotide.
Hanna rectifies this by teaching a segregating probe comprising a targeting moiety configured to form a complex with a rare species and a common species, a protective group, wherein the protective group is configured to provide a reduced rate of cleavage of a scissile bond in a first complex comprising the rare species and segregating probe relative to the rated cleavage of the scissile bond in a second complex comprising the common species and segregating probe (claim 11), wherein the rare species and the common species may be polynucleotides (claim 12).
Hanna teaches that segregating probe hybridized to the mismatched polynucleotide in which the cleavable portion of the probe is not protected is cleaved, whereas matched polynucleotide/segregating probe hybrid, which can be protected by minor groove binding remains intact (para [0086]).
Hanna teaches that the cleavage probes may be used with a variety of nucleic acid probe compositions including altered backbones including LNA and PNA and are compatible with at least Molecular Beacons, TaqMan probes, and Fret probes (para [00120]) and may be applied to a wide variety of nucleic acid detection/characterization technologies including PCR and RT-PCR (para [00119]; see also para [00122]). Hanna teaches suitable dyes include light emitting compounds (para [0079]).
Hanna teaches that a molecular species of interest may a be nucleic acid carrying a low frequency mutation, such as a SNP or small deletion or insertion relative or wild-type nucleic acid (para [0042]).
It is noted that the artisan would understand that the “wild type” and “mutant” species of probes of Hanna are a matter of nomenclature with regard to the structure of polynucleotides and could be switched as desired to suit the detection. See also MPEP 2144.04(IV).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute in the segregating probe of Hanna for the site-specific detection probe in the method of Princen. Such a substitution would have been obvious as the probes were taught as equivalents for the same purpose, given that the probes of Hanna encompass modified TaqMan, Molecular Beacons, and Fret probes, which are likewise are encompassed by the detector probes of Princen. See MEPE 2144.06(II). There would have been a strong expectation of success as both are directed to the detection of a rare polynucleotide target from a mixture that are not readily distinguishable due to nearly identical sequences, and such represents the application of a known product to a known technique.
It is noted that the 100-fold excess and signal-to-noise ratio also constitute matters of routine optimization. See MPEP 2144.05. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.
In the instant case, the instant specification teaches that a concentrations for the invention of “0.01 % and 0.001 % relative to corresponding wild type DNA” (para [0027]) and then later “mutation frequencies as low as 0.0001 %, 0.00003%, 0.00001 % or lower” (para [0070]). Likewise, the instant specification recites, “S/N ratio of 3.5, 5, 10 or greater is readily distinguishable from background and is readily detectable” (para [0064]). It is further noted that the instant specification recites that “robust detection (e.g. S/N> 15) [is possible] at mutation rates as low as 0.0003% relative to corresponding wild type DNA present in the sample” indicating that there is a link between the two values, further supporting these are a matter of optimization.
Regarding claim 2, in the method of Princen in view of Hanna, Princen teaches that modifications of bases and internucleotide linkages in oligonucleotides serving as probes (para [0143]). Lacking a definition in the specification, a “linker” is interpreted broadly to encompass such modifications of bases and/or internucleotide linkages in probes if it “coupled” in some way to a base of the probe “sequence” that is complementary to the second polynucleotide (any part thereof).
Hanna teaches a linker coupled to a base of a probe sequence complementary to the target polynucleotide (Fig. 5 and 11, para [0079]), and adding a mild alkaline sodium borate buffer to provide selective cleavage at the ester linkage (para [0081]), wherein preparing such a buffer comprises a step of adding concentrated nitric acid [i.e., an oxidizing agent] (para [00111]).
It is noted that changes to the order of steps would be obvious under MPEP 2144.04(IV)(C). In the instant case, there are no structures identified in the claims that currently require such an oxidizing agent to perform a recited step or function and thus require a particular sequence.
Regarding claim 3, in the method of Princen in view of Hanna, Princen teaches that the mass of the first and second polynucleotide may be less than 300 ng (Fig. 32C: 40, 100, and 20 ng reactions).
It is noted that such also constitutes a routine optimization. See MPEP 2144.05. In the instant case, Princen teaches detecting ranges of DNA (e.g., Fig. 32C) and the instant specification recites performing the method in amounts exceeding 300 ng (e.g., para [0061], [0064]).
Regarding claim 4-5 and 8, in the method of Princen in view of Hanna, Princen teaches that the mutation may be SNPs, insertions, or deletion mutations (para [0189]).
Regarding claims 9-10, in the method of Princen in view of Hanna, Princen teaches detecting at least a KRAS G12R mutation (Example 2, e.g., para [0299]; Fig. 3; Fig. 7).
Regarding claims 11-12, in the method of Princen in view of Hanna, Princen teaches detecting an EGFR L858R mutation (Example 2, e.g., para [0301]; Fig. 6).
Regarding claim 13-15, in the method of Princen in view of Hanna, Princen teaches that the polynucleotides may be at a ratio of less than about 1/1,000,000 (para [0184]).
It is noted that such also constitutes a routine optimization. See MPEP 2144.05. As discussed in the rejection of claim 1, the ratio is not identified as a critical value and appears to be linked to optimization with values of the signal-to-noise ratio.
Regarding claim 17, it is noted the term “clamping primer” lacks a definition in the specification and was not found to be a term of art. Princen teaches that a PNA-DNA duplex binds with greater strength, higher stability and more specificity than a DNA duplex (para [0205]) and that LNA modification increase binding affinity toward the complementary DNA (para [0203]). Such increased affinity/binding may be considered “clamping”. Thus, interpreted broadly, the claim encompasses application of any primer that includes PNA, LNA, or XNA.
In the method of Princen in view of Hanna, Princen teaches that the allele-specific primer comprises at least one nucleic acid modification (para [0252]), wherein the modification(s) is/are located at the 3’ end [i.e., a clamping primer] and the modification may be PNA or LNA or a combination thereof (para [0258]; see [0194] and Fig. 15 and 16).
For the sake of compact prosecution, is also noted that Princen also teaches allele-specific blocker probes, that may encompass one or more LNA modification (para [0222]).
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Princen (US 2014/0248612 A1; published 09/04/2014; as cited in the IDS dated 09/19/2024) in view of Hanna (WO 2019/165469 A1; published 08/29/2019) as applied to claim 1 above, and further in view of NEB (New England Bio Labs. Phusion DNA polymerase [Internet]. 2020 Apr 24 [cited 2026 Jan 4]. Available from: https://web.archive.org/web/20200424003201/https://www.neb-online.de/en/pcr-and-dna-amplification/high-fidelity-pcr/phusion-dna-polymerase/).
Regarding claim 16, in the method of Princen in view of Hanna, Princen teaches that various polymerase may be used (para [0241]) but fails to explicitly teach high fidelity polymerase.
NEB rectifies this by teaching Phusion DNA polymerase, and that it dramatically reduces extension times with increased yield (Advantages).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to utilize the Phusion DNA polymerase of NEB as the DNA polymerase in the method of Princen in view of Hanna, motivated by the desire to reduce extension times and/or increase yield, as taught by NEB. Such would have also been obvious as a substitution for the same purpose it was likewise taught for the same purpose of amplifying DNA in PCR. See MPEP 2144.06(II). There would have been a strong expectation of success as Princen and NEB are both directed to amplifying nucleic acids.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Kolpashchikov (US 9,121,053 B2; granted 09/01/2015) teaches SNP genotyping via amplification using specific primers (col 2, para 4) paired with a binary probe (col 2, para 4, spanning col 3), wherein the binary probes may use fluorescence or luminescence and detects target sequences at a ratio of 20:1 or greater [i.e., S/N ratio >=20] (col 6, para 2-3).
Kolpashchikov teaches a preferred embodiment where the binary DNA probe is a binary DNA peroxidase probe capable of binding hemin, wherein the complex of binary DNA peroxidase probe and hemin can catalyze oxidation of various substrates to luminescent products, which can be detected spectrophotometrically (col 6, para 7, spanning col 7). Kolpashchikov teaches binary DNA peroxidase probes—upon binding target sequences in the analyte—form a guanine quadruplex (G-quadruplex) that is capable of binding hemin, and that G-quadruplex-bound hemin demonstrates hydrogen peroxidase-like activity [i.e., may be considered an oxidizing agent] (col 6, para 7, spanning col 7).
Kolpashchikov teaches linkers in the probe coupled to the arm containing the SNP, wherein such arms are preferably 6-20 nucleotides (col 7, para 1-3).
Kolpashchikov doesn’t explicitly teach a fold excess of the rare polynucleotide species but describes that the amplification of the template nucleotide may be at least 3 to at least 10,000,000-fold greater than the starting DNA (col 4, para 6, spanning col 7).
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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636