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 .
Continued Examination Under 37 CFR 1.114
2. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on August 4, 2025 has been entered.
Claims 16-34 are pending and under examination.
Response to Arguments
3. Applicant’s arguments filed on June 27, 2025 and entered with the RCE submission of August 4, 2025 regarding the rejection of claims 16-34 under 35 U.S.C. 103 have been fully considered, but they were not persuasive.
Argument:
Applicant first argues that the references fail to teach or suggest probe with the required structural features (Remarks, page 6). More specifically, Applicant argues that Dietmaier fails to teach or suggest a probe with a target-complementary non-homopolymeric sequence at the 5’ and 3’ end of the homopolymeric sequence as required by all of the claims (Remarks, page 6). Applicant points to column 6, lines 43-44 and Figure 1 of Dietmaier to support this statement. Applicant additionally argues that only one portion of Dietmaier (i.e., col. 7, ll. 12-14) states that a probe may have more than one non-homopolymeric flanking region, and this portion of Dietmaier does not teach or suggest that both non-homopolymeric flanking regions are complementary to the target as required by all of the claims (Remarks, page 6). Applicant points to the next sentence in column 7 (i.e., at col. 7, ll. 15-18) and also column 5, lines 35-37 of Dietmaier to support this argument (Remarks, page 6). Response:
These arguments regarding Dietmaier have been fully considered, but they were not persuasive for essentially the same reasons set forth in the Advisory action mailed on July 9, 2025. These reasons are set forth again below with some additions and modifications to more fully address Applicant’s arguments.
The examiner agrees that the bulk of Dietmaier (e.g., Fig. 1 and the portion of column 6 cited by Applicant) relates to probes that have a non-repetitive target-complementary portion at the 5’ end OR the 3’ end. Nevertheless, the reference does explicitly state that a non-repetitive portion may be added to the 5’ end AND the 3’ end at column 7, lines 12-14. As discussed in MPEP 2123, the prior art is relevant for all that it contains, including nonpreferred or alternative embodiments. As well, as noted in the Final Rejection of April 4, 2025, Dietmaier is clearly not limited to probes with only one non-repetitive segment since column 6, line 43 uses open language by stating, “[T]he probe must comprise….”
Applicant is correct that the cited sentence of Dietmaier at column 7, lines 12-14 does not use the word “target-complementary” to describe the two non-repetitive sequences at the 5’ and 3’ end of the repetitive segment, but when the teachings of the reference as a whole are considered, it is clear that these two non-repetitive segments should be target-complementary segments. This is because the rest of Dietmaier describes 5’ or 3’ non-repetitive flanking segments as target-complementary (see, e.g., col. 6, penultimate paragraph and col. 7, lines 15-18). Therefore, when two non-repetitive segments are used as described in column 7, lines 12-14, it is clear that both should be target-complementary. Otherwise, they would not be described as “non-repetitive segments” by Dietmaier.
Applicant’s arguments at the bottom of page 6 regarding the disclosure of Dietmaier at column 7, lines 15-18 and column 5, lines 35-37 were also unpersuasive. The disclosure of Dietmaier at column 7, lines 15-18 apparently relates to the preferred embodiment in which the probe contains a target-complementary non-repetitive element at either the 5’ end or the 3’ end and not to the alternative embodiment in which the non-repetitive and target-complementary element is located at both ends. At column 5, lines 35-37, Dietmaier does disclose probes with 5’ or 3’ overhangs that do not hybridize to the target nucleic acid. It is not clear, though, that this does not simply represent an alternative embodiment in Dietmaier. Further, the ordinary artisan would have concluded that the non-repetitive sequences in a probe with 5’ and 3’ non-repetitive sequences should be designed to function in the same way as the non-repetitive sequences discussed elsewhere in Dietmaier (i.e., to be target-complementary).
Arguments:
Applicant also argues that the claimed probes can detect insertions and deletions, whereas “Dietmaier does not contemplate a probe having this advantage” (Remarks, page 7). Applicant points to col. 6, lines 57-63 to support this argument. Applicant additionally argues that the secondary references cited in the rejection (i.e., Knight, Oh, and Tyagi) do not remedy this deficiency in Dietmaier (Remarks, page 7).
Response:
These arguments were presented in the response preceding the Final Rejection mailed on April 4, 2025. The arguments have been reconsidered, but they remain unpersuasive for the reasons set forth previously.
Briefly, it is first noted that there is nothing in the claims that requires the probes to be capable of detecting insertions and deletions. Second, since the teachings of the references, particularly Dietmaier, suggest probes with the required structural features, the detection capabilities noted by Applicant in the Remarks are necessarily present. Still further, the teachings of Dietmaier at column 6, lines 57-63 apparently relate to the preferred embodiment, in which the probes have a target-complementary non-repetitive sequence at only one end, and not to the alternative embodiment, in which the probes have a target-complementary non-repetitive sequence at both ends. Lastly, since Dietmaier is not deficient with respect to this aspect of the probes, there is no deficiency for the secondary references to remedy.
Since Applicant’s arguments were not persuasive, the rejection has been maintained.
Claim Objections
4. Claims 28 and 29 are objected to because of the following minor informality. Each claim should be amended to insert the words “of the loop region” after “3’ end” in line 2 to more accurately describe the location of the non-homopolymeric sequence.
Claim Interpretation
5. It is noted that Applicant has provided explicit definitions for some of the terms recited in the claims. These definitions include the following:
(a) quantitative PCR or qPCR = simultaneously amplifying a detecting a target DNA molecule. In other words, the terms “quantitative PCR” and “qPCR” are synonymous with “real-time PCR.” See para. 24 on pages 7-8
(b) target homopolymeric nucleotide repeat sequence = the wild-type or reference homopolymeric repeat sequence as it is expected in the conditions where no MSI is present. See para. 28 on page 10.
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 16-34 are rejected under 35 U.S.C. 103 as being unpatentable over Dietmaier (US 6,664,064 B1) in view of Tyagi et al. (US 5,925,517 A), Knight et al. (US 2012/0052560 A1), and Oh et al. (The Journal of Molecular Diagnostics 2010; 12: 644-652).
Claims 16-26 are drawn to a cartridge. The cartridge includes the following elements: (a) a sample compartment for receiving a biological sample comprising a target nucleic acid; (b) a molecular beacon probe with a loop region that comprises a homopolymeric sequence; and (c) a polymerase with 3’-5’ exonuclease activity.
Claims 27-34 are drawn to a system that comprises the cartridge of claim 16 as well as a device comprising the following elements: (1) a cartridge receiver, (2) a thermocycler, (3) a fluorescence reader, and (4) software programmed to calculate a melting curve for a nucleic acid duplex formed by a molecular beacon probe hybridized to a target nucleic acid.
Dietmaier
Regarding claims 16-19 and 27-30, Dietmaier discloses probes useful for determining the number of repeat sequences in a target nucleic acid using melting curve analysis (abstract and col. 3, lines 11-26). The repeat sequence may be a homopolymeric repeat sequence of A, G, C, or T as recited in claims 19 and 30 and may have a length between 1-10 nucleotides (see, e.g., col. 4, line 66 - col. 5, line 14). This length for the repeat sequence overlaps with the recited length of 10-15 nucleotides recited in claims 16 and 27. The probes may be fluorescently labeled molecular beacon probes with a fluorophore at one end and a quencher at an opposite end that exhibit a change in fluorescence versus temperature during melting curve analysis (see, e.g., col. 6, lines 8-40; see also col. 1, line 65 - col. 2, line 7 and col. 7, lines 5-11 for additional discussion of the structural features of a molecular beacon). Dietmaier further teaches that the loop portion of the molecular beacon probes may contain a repeat-specific portion (i.e., a homopolymeric sequence complementary to the homopolymeric repeat sequence of interest) and a non-repetitive flanking sequence immediately 5’ and/or 3’ of the repeat-specific portion (col. 6, lines 41-63 and col. 7, lines 12-14). A non-repetitive flanking sequence may be at least three nucleotides in length (col. 6, lines 45-48). This length overlaps with the length ranges of 5-7 and 5-6 nucleotides recited in claims 17, 18, 28, and 29.
Regarding claims 23 and 26, Dietmaier discloses a homogeneous (i.e., “one-pot”) assay that uses a molecular beacon probe specific for a homopolymeric repeat sequence of interest, a polymerase, and a PCR primer pair (see, e.g., col. 6, lines 19-40).
Dietmaier does not teach all of the elements of the rejected claims. First, Dietmaier does not disclose a cartridge as required by claims 16 and 27. Second, Dietmaier is not anticipatory with respect to the specific lengths for the various features of the molecular beacon probes recited in claims 16-18 and 27-29. Third, Dietmaier fails to disclose using a polymerase having 3’-5’ exonuclease activity as required by independent claims 16 and 27.
Tyagi
Tyagi discloses molecular beacon probes, which may be unimolecular probes with the stem-loop structure recited in claim 16 (see, e.g., the abstract, Figure 3, and Example 1 at column 26, lines 15-67). As can be seen in Figure 3 and the accompanying discussion in Example 1, for example, the molecular beacon has an internal target-specific loop portion and a stem region that is located at the 5’ and 3’ ends of the molecule and hybridizes when the loop portion does not bind to a target nucleic acid. As a well, a fluorophore and quencher are located at opposite ends of the molecule. Tyagi teaches that the stem and loop regions in molecular beacon probes should be designed so that the stem remains hybridized in the absence of the target nucleic acid and separates when the loop hybridizes to the target nucleic acid (see, e.g., columns 9-11). This portion of Tyagi further notes that length influences the melting temperature of probe regions (see, in particular, col. 11). Tyagi also teaches that the stem region, which may be common to different beacons, may be 5-20 nucleotides in length (see, e.g., col. 19, lines 34-35). Tyagi also discusses appropriate label pairs at, e.g., columns 16-17 and further teaches that the disclosed probes may be used in real-time assays (see, e.g., col. 4, lines 5-8; col. 20, lines 23-49).
Knight
Neither Dietmaier nor Tyagi discusses a cartridge as recited in independent claims 16 and 27 or a system as recited in claim 27, but Knight discloses cartridge and system for conducting nucleic acid assays, including real-time PCR and melting analysis (abstract).
More specifically, Knight discloses a cartridge with the following features (see, e.g., Figs. 7-10 and the accompanying description in paras. 136-140):
(i) a sample compartment for receiving a biological sample containing a target nucleic acid as recited in claims 16 and 27 (i.e., storage wells 334, discussed in, e.g., para. 136 and shown in Fig. 7); and
(ii) a thermocycling qPCR compartment for conducting real-time PCR (i.e., PCR chip 328, which contains channels 364 that pass through thermal zone 350) (see, e.g., Fig. 9 and para. 138; see also paras. 108-109, 202, 218, and 222, which disclose real-time PCR).
Further regarding claims 24 and 27, the qPCR compartment in the cartridge of Knight necessarily has at least one transparent wall since it is described as useful for conducting real-time PCR assays with fluorescence detection (see, e.g., paras. 202 and 218-228).
Further regarding claims 25 and 27, the qPCR compartment in the cartridge of Knight is located downstream of the sample compartment to which it is fluidically connected (see, e.g., Fig. 7 and paras. 136-137).
Further regarding claim 27, Knight also discloses a system, with which the disclosed cartridge may be engaged (see, e.g., Figs. 4-5 and the accompanying description at paras. 131-133). The system includes the following components:
(i) a cartridge receiver with which the disclosed cartridge may be engaged (i.e., tray 314 discussed in paras. 132-133);
(ii) a thermocycler (i.e., cooling manifold connector assembly 306 described in para. 131 and further in paras. 120-125);
(iii) a fluorescence reader (i.e., optical system 310 described in para. 131; see also paras. 218-228); and
(iv) software capable of calculating a melting curve by monitoring fluorescence during denaturation of a double-stranded target nucleic acid in the presence of an intercalating dye or a sequence-specific probe (see paras. 111, 116, 128-130, 214-218, 225-226, 231, and 334).
Oh
Dietmaier, Tyagi, and Knight do not disclose a polymerase with 3’-5’ exonuclease activity, but Oh discloses a real-time PCR method conducted with molecular beacon probes and ExTaq DNA polymerase (page 646, “Real-Time PCR with the Molecular Beacon” section). As evidenced by the specification of the instant application at para. 32, the ExTaq DNA polymerase used by Oh has 3’-5’ exonuclease activity.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for the ordinary artisan in possession of the teachings of Dietmaier to design molecular beacon probes comprising the structural features of the loop region set forth in claims 16-18 and 27-29. As noted above, Dietmaier discloses a molecular beacon probe, labeled at one end with a fluorophore and at an opposite end with a quencher, that contains a loop region with a homopolymeric repeat sequence flanked by non-homopolymeric sequences complementary to the sequence immediately upstream and downstream of the homopolymeric repeat sequence. Dietmaier further discloses length ranges for the homopolymeric repeat sequence and non-homopolymeric flanking sequences that overlap with the claimed length ranges, and no evidence of unexpected results has been presented concerning these ranges. Accordingly, they are prima facie obvious per MPEP 2144.05 I.
It also would have been prima facie obvious to design molecular beacon probes with a stem region that is 5-7 nucleotides in length as recited in claims 16 and 27. Dietmaier does not discuss the length of the stem region of the disclosed beacons, but Tyagi renders the claimed length range obvious in view of the following: (1) Tyagi discloses an overlapping length range for the stem region in col. 19; (2) Tyagi additionally teaches designing the length of the stem and loop regions such that the unimolecular beacon probe only “opens” when the loop hybridizes to the target nucleic acid in, e.g., columns 9-11; and (3) no persuasive evidence of unexpected results has been presented with respect to the claimed length range. See also MPEP 2144.05 I.
It also would have been prima facie obvious to provide the molecular beacon probes suggested by Dietmaier in view of Tyagi in a cartridge together with the polymerase and primers disclosed by Dietmaier as suitable for use with molecular beacon probes and also to engage said cartridge with a system comprising the device set forth in claim 27. Knight provides motivation to do so by teaching that the disclosed systems and cartridges offer the ability to perform “rapid serial multiplex assays, on several samples simultaneously” (para. 9). The ordinary artisan also would have recognized that the cartridge and system disclosed in Knight would advantageously reduce the amount of “hands-on” time for users by automating steps in a method comprising amplification and melting curve analysis (e.g., pipetting) (see, e.g., paras. 12, 29, 37, and 181-182). Knight provides additional motivation by teaching that only small aliquots of a sample are required and that the disclosed systems offer flexibility to the user (para. 337). The ordinary artisan would have had a reasonable expectation of success since the teachings of Knight indicate that the disclosed cartridges and systems are broadly useful in the field of nucleic acid analysis and are not limited to any particular type of nucleic acid targets.
In providing the reagents suggested by Dietmaier and Tyagi in a cartridge as suggested by Knight, the ordinary artisan would have been particularly motivated to place the molecular beacon probe(s), the primers, and polymerase in the thermocycling qPCR compartment in the cartridge, recognizing that doing so would be necessary to perform the qPCR disclosed in Dietmaier (col. 6, lines 19-40). The ordinary artisan would have had a reasonable expectation of success since the qPCR compartment in the cartridge of Knight is suitable for real-time PCR (see, e.g., paras. 108-109, 111, 208, and 222). The ordinary artisan also would have been motivated to engage the cartridge with the system disclosed in Knight to obtain the ability to carry out an assay comprising real-time PCR and melting curve analysis.
Finally, it would have been prima facie obvious to select a polymerase with 3’-5’ exonuclease activity as the polymerase for inclusion in the thermocycling qPCR cartridge suggested by the references. As discussed in MPEP 2144.07, it is prima facie obvious to select a known material based on its suitability for the intended purpose in the absence of unexpected results. In this case, the teachings of Oh indicate that such a polymerase (ExTaq) can be used in real-time PCR compositions containing molecular beacons (page 646, “Real-Time PCR with the Molecular Beacon” section), and no evidence of unexpected results has been presented. Thus, the inclusion of a polymerase with 3’-5’ exonuclease in the cartridge suggested by the references is prima facie obvious.
Thus, the cartridges of claims 16-19 and 23-26 as well as the systems of claims 27-30 and 34 are prima facie obvious.
Further regarding claims 20-22 and 31-33, it also would have been prima facie obvious for the ordinary artisan to include at least two different molecular beacon probes, wherein the different molecular beacon probes have different homopolymeric sequences of different lengths and also different non-homopolymeric flanking sequences, in the cartridges suggested by the references. Dietmaier provides motivation to do so by teaching that probes may be designed for any desired homopolymeric repeat of interest and that a homopolymeric repeat sequence may be selected from A, G, C, or T (col. 4, line 67 - col. 5, line 9; col. 8, lines 24-43). Knight provides a reasonable expectation of success by teaching that the disclosed cartridges and systems are useful for multiplexed analysis (see, e.g., para. 9). Thus, the cartridges of claims 20-22 and the systems of claims 31-33 are prima facie obvious.
Conclusion
9. No claims are currently allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Angela Bertagna whose telephone number is (571)272-8291. The examiner can normally be reached 8-5, M-F.
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/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1637