DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. 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 the Application
2. Applicant’s response filed on July 9, 2025 has been entered.
Claims 1-16, 19-21, and 25 are pending. Claims 1-16 and 19-21 are under examination. Claim 25 remains withdrawn from consideration as being drawn to a nonelected invention.
Response to Arguments
3. Applicant’s arguments filed on July 9, 2025 have been fully considered.
Claim Objections
Applicant argues that the objections to claims 2-9, 11, 12, and 19-21 should be withdrawn in view of the claim amendments (Remarks, page 6).
This argument was persuasive. The objections have been withdrawn.
Rejection of claims 1-10, 15, and 16 under 35 U.S.C. 112(b)
Applicant argues that the rejection should be withdrawn in view of the amendments to claims 1, 2, 9, and 16 (Remarks, pages 6-7).
This argument was persuasive. The rejection has been withdrawn.
Rejection of claims 1-10 and 15 under 35 U.S.C. 103 as being unpatentable over Marras in view of Huang and Panousis
Applicant argues that the rejection should be withdrawn for the following three reasons: (i) the references fail to teach or suggest the combination of the first and second primers of claim 1 (Remarks, pages 7-9); (ii) the method of claim 1 exhibits unexpected results (Remarks, pages 9-10); and (iii) the claimed method addresses a long-felt need (Remarks, pages 10-11).
Argument (i):
Applicant argues that Marras discloses a common reverse primer rather than the allele-discriminating reverse primer required by independent claim 1 (Remarks, pages 7-8). Applicant then argues that neither Huang not Panousis remedies this deficiency in Marras because neither reference discloses an allele-discriminating multi-part primer (Remarks, pages 8-9). Therefore, Applicant argues, the ordinary artisan would have lacked motivation and a reasonable expectation of success in combining the teachings of the references to arrive at the claimed invention (Remarks, pages 8-9).
In response, the examiner agrees that Marras fails to disclose a reverse primer with the required structural features. Huang and Panousis are not alleged to teach or suggest the structural features of the allele-discriminating multi-part primer recited in claim 1, though. Instead, these references provide motivation for the ordinary artisan to substitute the common reverse primer used by Marras with an allele-discriminating multi-part primer having the same general structural features as the forward allele-discriminating multi-part primer disclosed by Marras. As discussed on page 10 of the last Office action, each of Huang and Panousis teaches that it can be useful, e.g., in the case when the target nucleic acid contains more than one SNP of interest, to amplify the target nucleic acid using a primer pair in which the forward and reverse primer are allele-discriminating (Huang at para. 12; Panousis at para. 156). The ordinary artisan would have recognized that when applying the teachings of Huang and Panousis concerning allele-discriminating primers to the method of Marras, forward and reverse allele-discriminating multi-part primers would be useful for this purpose. Thus, the references do, in fact, suggest use of the required primer pair.
As well, “A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton.” KSR, 550 U.S. at 421, 82 USPQ2d at 1397. And, “[I]n many cases, a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle.” Id. at 420, 82 USPQ2d at 1397. See MPEP 2141 II.C. This section of the MPEP also notes that “Office personnel may also take into account ‘the inferences and creative steps that a person of ordinary skill in the art would employ.’ Id. at 418, 82 USPQ2d at 1396.”
In this case, Marras discloses a method for amplifying a rare target nucleic acid that uses an allele-discriminating multi-part forward primer and a common reverse primer. Huang and Panousis each provide motivation to conduct the method of Marras using an allele-discriminating forward and reverse primer. In applying these teachings of Huang and Panousis to Marras, the ordinary artisan would have recognized, using ordinary creativity and inferences, that the forward and reverse allele-discriminating primers could be either (i) a forward allele-discriminating multi-part primer as taught in Marras and a conventional linear allele-discriminating reverse primer or (ii) multi-part forward and reverse allele-discriminating primers, each designed to have the multiple parts disclosed in Marras. Therefore, the ordinary artisan, using no more than the teachings of the references in combination with ordinary creativity, would have arrived at the primer pair used in the claimed method.
Argument (ii):
Applicant argues that the claimed method is associated with unexpected results (Remarks, pages 9-10). Here, Applicant argues that the claimed methods are highly sensitive, specific, and robust, pointing to para. 21 of the published application for support. Applicant also argues that the very high sensitivity of the claimed method results from the allele-discriminating and multi-part nature of the forward and reverse primers.
This argument was not persuasive for the following reasons. First, not all of Applicant’s arguments are commensurate in scope with the claimed invention. More specifically, the forward and reverse SuperSelective primers are only required to target the same mutant nucleotide in dependent claims 11-14. Similarly, targeting mutations that occur in cis is only required by dependent claims 19-21. As discussed in MPEP 716.02(d), evidence of unexpected results must be commensurate in scope with the claimed invention.
Second, it is not clear that the observed results are unexpected in view of the prior art since the cited McCoy reference teaches that the use of a forward and reverse primer, each of which is allele-discriminating for the same target mutation, can increase sensitivity (i.e., result in a lower LOD) (see, e.g., paras. 40 and 42). As discussed in MPEP 716.02(c), the results alleged to be unexpected must actually be unexpected.
Third, the fact that the inventors have recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In this case, the teachings of the secondary references (Huang, Panousis, and McCoy) provide a reason to use SuperSelective forward and reverse primers in the method of Marras. It is not clear from the arguments or the specification of the instant application that the observed sensitivity is unexpectedly greater than one might expect from following the teachings of the prior art to arrive at the claimed invention, particularly since Marras teaches that the general structural features of SuperSelective primers are associated with improved sensitivity (see, e.g., pp. 5-6 and 10-14).
Argument (iii):
Applicant argues that the claimed method satisfies a long-felt need in the art and points to the teachings in para. 5 of the pre-grant publication of the instant application (Remarks, pages 10-11).
This argument was not persuasive because the teachings in para. 5 of the pre-grant publication of the instant application do not satisfy the criteria for establishing that the claimed invention satisfies a long-felt need in the art. As discussed in MPEP 716.04, “Establishing long-felt need requires objective evidence that an art recognized problem existed in the art for a long period of time without solution.” This section of the MPEP goes on to state that the following factors must be considered when assessing long-felt need and the failure of others: (1) the need must have been persistent and recognized by ordinary artisans; (2) the need must not have been satisfied by others prior to the claimed invention; and (3) the invention must, in fact, satisfy the long-felt need.
In this case, the statements in para. 5 of the pre-grant publication of the instant application does not constitute the objective evidence discussed in MPEP 716.04 because it is not accompanied by any supporting evidence (e.g., review or other non-patent literature articles or evidence discussing the problem). Instead, the statements in para. 5 function essentially as an unsupported opinion, which is insufficient to establish that the problem existed in the art for a long time and remained unaddressed. There is also nothing in para. 5 that would satisfy factors (1) and (2) above. Lastly, the cited prior art of Marras appears to address the alleged long-felt need because that reference teaches that the disclosed SuperSelective primers offer highly sensitive, rapid, and noninvasive detection without the need for novel thermocyclers (see, e.g., pp. 5-8, 12, 14, and 36-42, and 46-47).
Since Applicant’s arguments were not persuasive, the rejection has been maintained with minor modifications to address the removal of the term “closely related sequence” and the use of “unintended target sequence” throughout the claims.
Rejections of claims 16 and 19-21 under 35 U.S.C. 103 citing Marras, Huang, and Panousis as the primary combination of references
Applicant argues that the rejection should be withdrawn because the secondary references cited in the rejections fail to remedy the deficiencies in the primary combination of references (Remarks, pages 11-12).
This argument was not persuasive because the primary combination of references is not deficient for the reasons set forth above. The rejections have been maintained.
Rejection of claims 1-15 under 35 U.S.C. 103 as being unpatentable over Marras in view of McCoy
Applicant argues that the rejection should be withdrawn for essentially the same reasons set forth above with respect to the rejection of claims 1-10 and 15 under 35 U.S.C. 103 citing Marras, Huang, and Panousis (Remarks, pages 12-13). Here, Applicant reiterates the arguments concerning the teachings of Marras, unexpected results, and long-felt need and also argues that McCoy fails to remedy the deficiencies of Marras because the reference fails to teach or suggest the structure of the allele-discriminating multi-part primer of claim 1.
Applicant’s arguments concerning unexpected results, long-felt need, and the teachings of Marras were unpersuasive for the reasons set forth above in response to the rejection of claims 1-10 and 15 under 35 U.S.C. 103 citing Marras, Huang, and Panousis.
Applicant’s arguments concerning McCoy were also unpersuasive because that reference is not alleged to teach or suggest the structural features of the allele-discriminating multi-part primer recited in claim 1. Rather, McCoy is cited to provide motivation for the ordinary artisan to substitute the common reverse primer used by Marras with an allele-discriminating multi-part primer having the same general structural features as the forward allele-discriminating multi-part primer disclosed by Marras. As discussed in the rejection on page 17, McCoy teaches that the use of allele-discriminating forward and reverse primers can be useful when designing an allele-specific assay since they resulting assay may have a lower LOD. The ordinary artisan would have recognized that when applying the teachings of McCoy concerning allele-discriminating primers to the method of Marras, forward and reverse allele-discriminating multi-part primers would be useful for this purpose. Thus, the teachings of the references do, in fact, suggest a primer pair with the required structural characteristics.
And, the discussion of KSR set forth above with respect to the rejection of claims 1-10 and 15 under 35 U.S.C. 103 citing Marras, Huang, and Panousis applies here as well.
Since Applicant’s arguments were not persuasive, the rejection has been maintained with minor modifications to address the removal of the term “closely related sequence” and the use of “unintended target sequence” throughout the claims.
Claim Interpretation
4. The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The following is a quotation of pre-AIA 35 U.S.C. 112, sixth paragraph:
An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph:
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action.
In independent claim 1, the term “homogeneous fluorescence detection means” is used in step (a), line 3 has been treated under 35 U.S.C. 112(f).
The specification teaches that “homogeneous fluorescence detection means” include the following: (i) homogeneous fluorescence detection probes (e.g., an interprimer-specific molecular beacon probe) (page 4); (ii) a molecular beacon probe, a TaqMan probe, a minor groove binder (MGB) probe, or an MNAzyme/cleavable probe combination (page 10); (iii) a labeled primer (e.g., a Scorpion or LUX primer) (page 10); and (iv) an intercalating dye (e.g., SYBR® Green) (page 10).
Claim Objections
5. Claim 1 is objected to for the following reason. Amending the claim to replace all instances of “first forward primer” and “second reverse primer” with “forward primer” and “reverse primer,” respectively, is suggested to avoid creating the impression that two reverse primers are used.
Claims 2, 3, 11, 12, 14, and 19, which depend from claim 1 and also recite “first forward primer” and/or “second reverse primer,” are objected to for the same reason set forth above.
Claim Rejections - 35 USC § 103
6. 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.
7. 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.
8. Claims 1-10 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Marras et al. (WO 2017/176852 A1; cited previously) in view of Huang et al. (US 2013/0323727 A1; cited previously) and Panousis et al. (US 2016/0258010 A1; cited previously).
The instant claims are drawn to a method for amplifying and detecting a rare target nucleic acid. The method uses allele-discriminating primers.
Regarding claims 1, 2, and 5, Marras discloses a method for amplifying and detecting rare target sequences present in samples containing large amounts of unintended target sequences (see, e.g., page 5, line 12 – page 6, line 15). Marras notes that the disclosed methods are capable of detecting as few as ten copies of a rare target DNA sequence in a background of 10,000 copies of an unintended target sequence (page 5, lines 12-24). More specifically, the method of Marras includes the following steps:
(a) preparing a primer-dependent amplification reaction mixture that includes a sample; a DNA polymerase; dNTPs; an amplification buffer; homogeneous fluorescence detection means for detecting amplification products; and a primer pair for each rare target sequence, wherein the first primer in the pair is a SuperSelective primer that is specific for the rare target sequence and mismatched to the unintended target sequence;
(b) performing a non-symmetric PCR to amplify the rare target sequence; and
(c) detecting the rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means.
See page 5, lines 12-29; page 6, lines 5-15; and page 14, lines 8-26. Marras further discusses the homogeneous detection means at, for example, page 12, lines 21-28. As well, the SuperSelective primers used in the method of Marras have the 5’ anchor sequence, internal bridge sequence, and 3’ foot sequence that is mismatched to the unintended target sequence at least at its 3’-terminal or 3’-penultimate nucleotide required by claim 1 (see Marras at page 10, lines 1-32 and page 13, lines 14-22).
Regarding claims 3 and 4, as noted above, Marras teaches the use of a SuperSelective primer as one of the primers in the primer pair (page 14, lines 8-19). Such a primer may have a 3’-terminal or 3’-penultimate interrogating nucleotide that is complementary to the rare target sequence and mismatched to the unintended target nucleic acid (see, e.g., page 13, lines 14-22).
Regarding claim 6, Marras teaches that the detecting in the non-symmetric PCR may be real-time detecting (see, e.g., page 14, 8-26).
Regarding claim 7, Marras teaches that the PCR may be a digital PCR and that detection may be end-point detection (see, e.g., claims 1 and 9; see also page 8, lines 10-15; page 9, lines 20-25; and page 20, line 11 – page 21, line 3).
Regarding claims 8-10, Marras teaches that the sample may include at least two different rare target nucleic acids and that the method may comprise the use of a different homogeneous fluorescent detection probe for each different rare target sequence (see, e.g., page 6, line 20 – page 7, line 12). The different homogeneous fluorescence detection probes may be color-coded (i.e., labeled with different fluorophores) (page 6, line 20 – page 7, line 12; page 8, lines 10-18) or may contain the same fluorescent label and be differentiated by Tm differences (page 7, lines 13-17; page 15, lines 2-5; page 21, lines 4-8).
Regarding claim 15, Marras teaches that the fluorescent detection probes may hybridize to the bridge sequence or its complement (see, e.g., page 31, lines 15-21; page 32, lines 3-9; page 56, line 29 – page 57, line 12).
Marras does not teach all of the elements of the rejected claims. As to claim 1, since Marras teaches a common reverse primer (see, e.g., page 14, lines 8-14), the reference fails to teach a primer pair for a rare target nucleic acid in which both primers in the pair are allele-discriminating primers (e.g., with each primer having a 3’-terminal interrogating nucleotide that is complementary to the rare target nucleic acid but not to the unintended target nucleic acid as recited in claim 4). Marras also fails to teach that both primers in the primer pair are multi-part primers, with the second primer in the pair being a SuperSelective primer, as required by claim 3.
Prior to the effective filing date of the claimed invention, though, it would have been prima facie obvious to practice the method of Marras using an allele-discriminating second primer as required by the instant claim 1. Huang and Panousis each provide motivation to do so by teaching that it can be desirable in some situations to use an allele- discriminating forward primer and an allele- discriminating reverse primer to amplify a target nucleic acid of interest (Huang at, e.g., para. 12; Panousis at, e.g., para. 156). More specifically, the cited portion of each reference teaches that an allele- discriminating forward and reverse primer can be used to detect the simultaneous presence of multiple SNPs of interest on a target nucleic acid. The ordinary artisan practicing the method of Marras would have been motivated to use a forward and reverse allele-discriminating primer to obtain this ability to detect the presence of multiple SNPs in a target nucleic acid in a single reaction and would have had a reasonable expectation of success in view of the guidance provided throughout the references concerning the design of allele-discriminating primers. Thus, the methods of claims 1, 2, 5-10 and 15 are prima facie obvious.
Further regarding claim 3, it also would have been prima facie obvious to use forward and reverse SuperSelective primers, each of which is mismatched to the unintended target sequence by at least the 3’-terminal nucleotide or the 3’-penultimate nucleotide, when practicing the method suggested by Marras in view of Huang and Panousis. The ordinary artisan would have been motivated to do so since Marras taught that SuperSelective primers are “both ‘specific’…and highly ‘selective’ (page 3, lines 30-31) and would have had a reasonable expectation of success since Marras provides extensive guidance as to the design of such primers on pages 10-13 and there is nothing in the reference to suggest that pairs of SuperSelective primers cannot be used to amplify a desired target nucleic acid. Thus, the method of claim 3 is also prima facie obvious.
Further regarding claim 4, when the method is practiced using a SuperSelective forward and reverse primer, each such primer may have the required 3’-terminal interrogating nucleotide since Marras teaches that SuperSelective primers may have a 3’-terminal or 3’-penultimate interrogating nucleotide (page 13, lines 15-22). Thus, the method of claim 4 is also prima facie obvious.
9. Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Marras et al. (WO 2017/176852 A1; cited previously) in view of Huang et al. (US 2013/0323727 A1; cited previously) and Panousis et al. (US 2016/0258010 A1; cited previously) and further in view of Tsourkas et al. (Nucleic Acids Research 2002; 30: 4208-4215; cited previously).
As discussed above, the teachings of Marras in view of Huang and Panousis render obvious the methods of claims 1-10 and 15.
Marras teaches the use of molecular beacon probes (see, e.g., page 56, line 29 – page 7, line 30), but fails to teach that the probes are shared-stem molecular beacons as required by claim 16.
Prior to the effective filing date of the claimed invention, though, it would have been prima facie obvious for the ordinary artisan to substitute the molecular beacons used in the method suggested by Marras in view of Huang and Panousis with shared-stem molecular beacons. Tsourkas provides motivation to do so by teaching that such molecular beacons “form more stable duplexes with target molecules than conventional molecular beacons” (abstract). Tsourkas provides additional motivation by teaching that the design of shared stem beacons “helps immobilize the fluorophores of molecular beacons when they hybridize to the target, which is desirable when two molecular beacons are used in a FRET assay” (page 4214, column 1) and also noting that shared-stem beacons have “a shorter loop length, thus reducing the potential for secondary structure within the loop portion of the beacon” (page 4215, column 1). The ordinary artisan would have been motivated to use shared-stem beacons in the methods suggested by Marras in view of Huang and Panousis to obtain these advantages and would have had a reasonable expectation of success since Tsourkas provides guidance as to the design of shared-stem molecular beacons (page 4209). Thus, the method of claim 16 is prima facie obvious.
10. Claims 19-21 are rejected under 35 U.S.C. 103 as being unpatentable over Marras et al. (WO 2017/176852 A1; cited previously) in view of Huang et al. (US 2013/0323727 A1; cited previously) and Panousis et al. (US 2016/0258010 A1; cited previously) and further in view of Uchibori et al. (Nature Communications 2017; 8: 14768; cited previously).
As discussed above, the teachings of Marras in view of Huang and Panousis render obvious the methods of claims 1-10 and 15.
Regarding claim 19, these references do not teach applying the method to target nucleic acids in which the rare target nucleic acid differs from the unintended target nucleic acid by a first base pair and a second base pair that occur in cis, and performing the amplification reaction using first primer that is complementary to the first base pair and a second primer that is complementary to the second base pair.
Prior to the effective filing date of the claimed invention, though, it would have been prima facie obvious to practice the method suggested by Marras in view of Huang and Panousis using a rare target nucleic acid and primers as recited in claim 19. As discussed above, the teachings of Marras in view of Huang and Panousis suggest the use of forward and reverse allele-discriminating primers. These references do not teach that the mutations detected by these primers occur in cis, but Uchibori teaches that “[W]hen the C797S and T790M mutations [in the EGFR gene] developed in cis, all sensitivity to any of the existing EGFR-TKIs, including the third-generation ones, was lost” (page 1, col. 2). The ordinary artisan would have recognized from the teachings of Uchibori that detecting the presence of the C797S and T790M mutations in cis would be desirable since it would allow one to avoid selecting an ineffective cancer treatment. The ordinary artisan would have had a reasonable expectation of success since Marras, Huang, and Panousis provide guidance as to the design of allele-discriminating primers and the two mutations in question are relatively close to one another on the EGFR gene. Thus, the method of claim 19 is prima facie obvious.
Further regarding claim 20, which depends from claim 19, as discussed above, Marras teaches applying the method to a plurality of rare target nucleic acids and using a different homogeneous fluorescent detection probe for each different rare target nucleic acid (see, e.g., page 6, line 20 – page 7, line 17 and page 8, lines 10-18). Thus, the method of claim 20 is also prima facie obvious.
Further regarding claim 21, which depends from claim 19, the teachings of Marras also suggest detecting amplification products using an interprimer-specific molecular beacon probe for each different rare target nucleic acid. In particular, Marras teaches the use of molecular beacons generally (see, e.g., page 4, lines 27-30) and also notes that a preferred embodiment comprises targeting the beacon to a 5’ tag sequence on a SuperSelective primer (see, e.g., page 6, lines 20-30). The ordinary artisan would have recognized from these teachings of Marras that the molecular beacon probes for each different rare target nucleic acid could be either conventional molecular beacons, which are known to hybridize to the target nucleic acid between the regions targeted by the primers, or the 5’-tag-targeting molecular beacons discussed in more detail in Marras, and accordingly, would have been motivated to select either option with a reasonable expectation of success, especially when the method is designed to amplify and detect only a small number of different rare target nucleic acids. Thus, the method of claim 21 is also prima facie obvious.
11. Claims 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over Marras et al. (WO 2017/176852 A1; cited previously) in view of McCoy et al. (US 2014/0274786 A1; cited previously).
The instant claims are drawn to a method for amplifying and detecting a rare target nucleic acid. The method uses allele-discriminating primers.
Regarding claims 1, 2, and 5, Marras discloses a method for amplifying and detecting rare target sequences present in samples containing large amounts of unintended target sequences (see, e.g., page 5, line 12 – page 6, line 15). Marras notes that the disclosed methods are capable of detecting as few as ten copies of a rare target DNA sequence in a background of 10,000 copies of an unintended target sequence (page 5, lines 12-24). More specifically, the method of Marras includes the following steps:
(a) preparing a primer-dependent amplification reaction mixture that includes a sample; a DNA polymerase; dNTPs; an amplification buffer; homogeneous fluorescence detection means for detecting amplification products; and a primer pair for each rare target sequence, wherein the first primer in the pair is a SuperSelective primer that is specific for the rare target sequence and mismatched to the unintended target sequence;
(b) performing a non-symmetric PCR to amplify the rare target sequence; and
(c) detecting the rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means.
See page 5, lines 12-29; page 6, lines 5-15; and page 14, lines 8-26. Marras further discusses the homogeneous detection means at, for example, page 12, lines 21-28. As well, the SuperSelective primers used in the method of Marras have the 5’ anchor sequence, internal bridge sequence, and 3’ foot sequence that is mismatched to the unintended target sequence at least at its 3’-terminal or 3’-penultimate nucleotide required by claim 1 (see Marras at page 10, lines 1-32 and page 13, lines 14-22).
Regarding claims 3 and 4, as noted above, Marras teaches the use of a SuperSelective primer as one of the primers in the primer pair (page 14, lines 8-19). Such a primer may have a 3’-terminal or 3’-penultimate interrogating nucleotide that is complementary to the rare target sequence and mismatched to the unintended target nucleic acid (see, e.g., page 13, lines 14-22).
Regarding claim 6, Marras teaches that the detecting in the non-symmetric PCR may be real-time detecting (see, e.g., page 14, 8-26).
Regarding claim 7, Marras teaches that the PCR may be a digital PCR and that detection may be end-point detection (see, e.g., claims 1 and 9; see also page 8, lines 10-15; page 9, lines 20-25; and page 20, line 11 – page 21, line 3).
Regarding claims 8-10 and 13, Marras teaches that the sample may include at least two different rare target nucleic acids and that the method may comprise the use of a different homogeneous fluorescent detection probe for each different rare target sequence (see, e.g., page 6, line 20 – page 7, line 12). The different homogeneous fluorescence detection probes may be color-coded (i.e., labeled with different fluorophores) (page 6, line 20 – page 7, line 12; page 8, lines 10-18) or may contain the same fluorescent label and be differentiated by Tm differences (page 7, lines 13-17; page 15, lines 2-5; page 21, lines 4-8).
Regarding claim 14, Marras teaches that the disclosed Superselective primers may include a 5’ tag sequence, the complement of which is the target of the detection probe (see, e.g., Figures 1-2; page 6, line 20 – page 7, line 12; and page 12, lines 17-28).
Regarding claim 15, Marras teaches that the fluorescent detection probes may hybridize to the bridge sequence or its complement (see, e.g., page 31, lines 15-21; page 32, lines 3-9; page 56, line 29 – page 57, line 12).
Marras does not teach all of the elements of the rejected claims. As to claim 1, since Marras teaches a common reverse primer (see, e.g., page 14, lines 8-14), the reference fails to teach a primer pair for a rare target nucleic acid in which both primers in the pair are allele-discriminating primers (e.g., with each primer having a 3’-terminal interrogating nucleotide that is complementary to the rare target nucleic acid but not to the unintended target nucleic acid as recited in claim 4). Marras also fails to teach that both primers in the primer pair are multi-part primers, with the second primer in the pair being a SuperSelective primer, as required by claim 3. Lastly, since Marras fails to teach a pair of primers for a rare target nucleic acid in which both primers in the pair are allele-discriminating primers, the reference also fails to meet all of the requirements of claims 11, 12, and 14, which further limit the structure of these two allele-discriminating primers.
Prior to the effective filing date of the claimed invention, though, it would have been prima facie obvious for the ordinary artisan to practice the method of Marras using first and second primers that are each mismatched to the same single base pair in a rare target nucleic acid sequence (i.e., two allele-discriminating primers as recited in claim 1). As discussed above, Marras teaches the use of one allele-discriminating primer (e.g., a SuperSelective primer) in combination with a primer that is not an allele-discriminating primer. Marras also teaches that the rare target sequence may differ from the unintended target sequence by only a single base pair (see, e.g., page 5, lines 12-17). Marras does not teach or suggest that the first and second primers are mismatched to the single base pair, but McCoy provides motivation and a reasonable expectation of success by teaching that this is an acceptable way to design an allele-specific assay (para. 169; see also paras. 152-154 and Fig. 9). McCoy provides additional motivation by teaching that such assays can lower the LOD (paras. 40 and 42). Therefore, combining the teachings of Marras and McCoy results in a method in that uses two allele-discriminating primers, one of which is a SuperSelective primer, to detect a rare target nucleic acid. Thus, the methods of claims 1, 2, 5-11, and 13-15 are prima facie obvious.
Further regarding claims 3 and 12, it also would have been prima facie obvious to use forward and reverse SuperSelective primers, each of which is mismatched to the unintended target sequence by at least the 3’-terminal nucleotide or the 3’-penultimate nucleotide, when practicing the method suggested by Marras in view of McCoy. The ordinary artisan would have been motivated to do so since Marras taught that SuperSelective primers are “both ‘specific’…and highly ‘selective’ (page 3, lines 30-31) and would have had a reasonable expectation of success since Marras provides extensive guidance as to the design of such primers on pages 10-13 and there is nothing in the reference to suggest that pairs of SuperSelective primers cannot be used to amplify a desired target nucleic acid. Further regarding claim 12, it is noted that SuperSelective primers contain the required 5’ anchor sequence, internal bridge sequence, and 3’ foot sequence (see, e.g., pages 12-13 of Marras). Thus, the methods of claims 3 and 12 are also prima facie obvious.
Further regarding claim 4, when the method is practiced using a SuperSelective forward and reverse primer, each such primer may have the required 3’-terminal interrogating nucleotide since Marras teaches that SuperSelective primers may have a 3’-terminal or 3’-penultimate interrogating nucleotide (page 13, lines 15-22). Thus, the method of claim 4 is also prima facie obvious.
Conclusion
12. No claims are currently allowable.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1681