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 .
Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Interview Summary
Applicant’s submission of an interview summary on 1/27/2026 regarding the interview conducted on 1/7/2026 is acknowledged.
Claim Status
Claims 1-2, 6, 9, 40-44, and 46-54 are pending and being examined on the merits.
Specification
Withdrawn:
The objection to the specification for informalities is withdrawn in light of Applicant’s amendments to paragraph [0012]. Applicant’s amendment to the specification to properly denote trade names or marks used in commerce is acknowledged.
The objection to the specification for failing to provide proper antecedent basis for the claimed subject matter is withdrawn in light of Applicant’s amendments to the claims.
New (Necessitated by Amendments):
The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required: “the fluorescent probe comprises a lower melting temperature in relation to the nucleic acid molecule compared to a melting temperature of the probe complement in relation to the nucleic acid molecule” is a limitation in the present claims which does not find support in the disclosure (as covered below in the 112a rejection).
Please note that the above claim limitations have been rejected under 112a as being “new matter” added to the claims (see below). Applicant is cautioned against amending the specification in any way that may introduce new matter into the disclosure.
Response to Remarks
Applicant argues that the objection to the specification for not providing proper antecedent basis for the claims is moot in light of the amendments to the claims. As noted in the new 112a rejection below, the amendment to claim 54 also constitutes new matter, therefore warranting a new objection to the specification for not providing proper antecedent basis for the limitation of claim 54.
Claim Objections
The objection to claim 9 is withdrawn in light of Applicant’s amendment to the claim.
Claim Rejections - 35 USC § 112a – New Matter
Withdrawn:
The rejection of claims 1-2, 6, 9, 40-54 under 35 U.S.C. 112(a) is withdrawn in light of Applicant’s amendments to the claims and cancellation of claim 45.
New (Necessitated by Amendments):
Claim 54 is 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. This is a new matter rejection.
Claim 54 contains the limitation “wherein the fluorescent probe comprises a lower melting temperature in relation to the nucleic acid molecule compared to a melting temperature of the probe complement in relation to the nucleic acid molecule”. This limitation is not present in the claim set as filed on 6/9/2022 and does not find adequate written support in the specification. The specification only discusses the potential melting temperature ranges for the probe complement in relation to the fluorescent probe, and only mentions a melting temperature of the fluorescent probe in relation to the nucleic acid molecule as being higher than that of the probe complement in relation to the fluorescent probe (“In some embodiments, a melting temperature of the complementary nucleic acid and the region of the probe nucleic acid is lower than a melting temperature of the probe nucleic acid and the target nucleic acid”; paragraph [0023]). Therefore, the limitation of claim 54 constitutes addition of new matter and lacks adequate written description in the disclosure.
Response to Remarks
Applicant’s amendment to claim 54 in response to the 112a New Matter rejection does not overcome the rejection, but introduces a new claim limitation which also constitutes new matter. The disclosure as filed does not mention any melting temperatures of the complementary nucleic acid (probe complement) in relation to the nucleic acid molecule.
Claim Rejections - 35 USC § 112b - Indefiniteness
Withdrawn:
The rejection of claims 1-2, 6, 9, and 40-54 under 35 U.S.C. 112(b) as described in the Office Action of 10/01/2025 is withdrawn in light of Applicant’s amendments of the claims and cancellation of claim 45.
New (Necessitated by Amendments):
Claim 46 and 54 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 46 depends from a cancelled claim (claim 45) and as such the scope of the claim is indefinite. For the purposes of examination, claim 46 is being interpreted as depending from claim 1, but clarification is required.
Claim 54 recites the limitation “wherein the fluorescent probe comprises a lower melting temperature in relation to the nucleic acid molecule compared to a melting temperature of the probe complement in relation to the nucleic acid molecule”. It is unclear how the probe complement could have a melting temperature in relation to the nucleic acid molecule given that both the probe complement and the nucleic acid molecule are the complements of the fluorescent probe and therefore would not be able to hybridize together. In the interest of customer service and compact prosecution, for the purposes of examination this claim is being interpreted to mean that the melting temperature between the fluorescent probe and the complement probe is lower than the melting temperature between the fluorescent probe and the nucleic acid molecule. This interpretation is consistent with the instant specification (see paragraph [0023]). However, further clarification is required.
Claim Rejections - 35 USC § 103
Withdrawn:
The rejection of claims 1-2, 6, 9, and 40-54 under 35 U.S.C. 103 as described in the Office Action of 10/01/2026 is withdrawn in light of Applicant’s amendments to the claims and cancellation of claim 45.
New (Necessitated by Amendments):
As noted in the 112b rejection of claim 46 above, for the purposes of examination claim 46 is being interpreted as depending from claim 1 (instead of cancelled claim 45).
Claims 1, 2, 6, 40-44, 46-48, and 52 are rejected under 35 U.S.C. 103 as being unpatentable over Regan (Regan et al., WO 2015/023677 A1; cited on IDS of 8/27/2025) in view of Fei (Fei et al., WO 2020/001529 A1; EFD of June 7, 2018; cited on IDS of 4/4/2025).
Regarding claims 1 and 2: Regan teaches a method for detection of amplified nucleic acid products in a plurality of partitions through the use of an oligomer reporter system. The oligomer reporter system is comprised of two probes (or oligomers), a reporter probe (reads on fluorescent probe) and a complementary probe (reads on probe complement; Abstract and pg 2, ln 20-33). Regan teaches formation of a plurality of partitions, in which a target (nucleic acid molecule) is contacted with a polymerase, probe (fluorescent probe), and sink (probe complement; pg 13, ln 25-28). The plurality of partitions are aqueous partitions surrounded by an immiscible fluid (pg 23, ln 1-20). The probe is complementary to (anneals to) the target nucleic acid molecule and the sink is complementary to the probe (Fig 1-3). Regan teaches a methodology in which the annealing of the sink to the probe reduces the photoluminescence of the reporter probe and thereby reduces “photoluminescent background of partitions that do not contain the product of interest” (reads on thereby reducing a background fluorescence of the partition; pg 2, ln 23-25 and pg 7, ln 6-8). Regan teaches that the probe complement (sink) comprises a first quencher (pg 10, ln 17-21 and Fig 1-2). Regan teaches, after contacting the target nucleic acid molecule with a polymerase, probe, and probe complement, amplifying the target nucleic acid (Fig 3, pg 6-7), and then imaging the partitions to detect fluorescence in partitions with successful amplification of a target nucleic acid (pg 17).
Regan does not explicitly teach that the partitions formed are in a three-dimensional distribution and does not teach imaging with a three-dimensional (3D) imaging technique (claim 1a and d) or that the 3D imaging technique is light sheet imaging (claim 2). However, use of light sheet imaging to image a plurality of partitions in a 3D distribution within a tube to detect fluorescence of amplification reactions is known in the art, as taught by Fei.
Fei teaches a method of 3D imaging of emulsified droplets using light sheet fluorescence microscopic imaging (Abstract). Fei teaches creating emulsified droplets which contain the necessary reaction components for a biochemical reaction such as a digital chain reaction (such as dPCR) and imaging said droplets (paragraphs [0019-0023]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Regan with that of Fei. One would be motivated to perform 3D imaging using the light sheet imaging technique of Fei given the assertion by Fei that this imaging setup allows for parallel high-throughput detection to be performed (paragraph [0026]). Additionally, Fei teaches that light sheet imaging of a 3D distributed sample allows for great utilization of the energy rate of laser light (paragraph [0024]). One would have a reasonable expectation of success given that Fei successfully uses light sheet imaging to image a 3D distribution of emulsified droplets and detect fluorescent results of an amplification reaction (paragraphs [0076-0094]).
Regarding claim 6: Regan teaches a probe complement that lacks a string of at least four sequential guanines (sink: 5’-CGCCCCTCAGATA-IB-3’; pg 37, ln 9-19).
Regarding claims 40-41: Regan teaches that the fluorescent probe (reporter probe) comprises a fluorophore and that this fluorophore is at the 5’ end of the fluorescent probe (pg 9, ln 22-24, pg 10, ln 5-6, and Fig 1-2).
Regarding claims 42-44: Regan teaches that the fluorescent probe may also comprise a second quencher attached to the 3’ end of the fluorescent probe (pg 9, ln 22-24 and Fig 1-2). Regan teaches that the quencher “may be configured to reduce an ability of photoluminophore 60 to emit light when excited” by “quenching emission of light from the photoluminophore” (pg 11, ln 22-28). This reads on “the second quencher comprises an absorption spectrum that overlaps with an emission spectrum of a fluorophore of the fluorescent probe”.
Regarding claim 46: Regan teaches that the probe complement (sink) comprises a quencher at the 3’ end (pg 10, ln 17-21 and Fig 1-2).
Regarding claim 47: Regan teaches that the probe complement comprises a mismatch relative to a nucleic acid sequence of the fluorescent probe (Fig 23 and pg 38, ln 10-34 and pg 39, ln 1-21).
Regarding claim 48: Regan teaches that the DNA polymerase used can have nuclease activity (e.g., Taq DNA polymerase; pg 12, ln 1-3).
Regarding claim 52: Regan teaches that the fluorescent probe comprises 15-25 nucleotides (Example 12, the probes 52a and 52b are 26 nucleotides long; pg 38, ln 29-32). The claim language of “comprises 15-25 nucleotides” indicates that the probe can contain at least 15 nucleotides and gives no upper limit for how many nucleotides the probe may comprise. Given this interpretation, a 26 nt probe “comprises” 25 nucleotides.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over (Regan et al., WO 2015/023677 A1; cited on IDS of 8/27/2025) in view of Fei (Fei et al., WO 2020/001529 A1; EFD of June 7, 2018; cited on IDS of 4/4/2025) as applied to claims 1, 2, 6, 40-44, 46-48, and 52 above, and further in view of Abate (Abate et al., WO 2019/139650 A2; cited on PTO-892 of 10/01/2025).
The teachings of Regan in view of Fei are detailed above. Relevant to the instantly rejected claims, Regan in view of Fei teach partitioning target nucleic acids and reaction components into aqueous partitions within an immiscible fluid.
Regan teaches that the partitions may contain the nucleic acid target at "partial occupancy", wherein one or more do not contain at least one copy of the target and only a subset contain at least one copy (pg 15). Regan in view of Fei do not teach that about 10% of the partitions comprise zero or one nucleic acid molecule. However, distribution of zero or one target nucleic acid molecules into aqueous partitions in an immiscible fluid is known in the art, as taught by Abate.
Abate teaches a method of generating monodisperse iemulsions that encapsulate targets of interest before performing reactions such as ddPCR (Abstract and Fig 3). Abate teaches that the monodisperse emulsions are generated such that the droplets contain on of the monodisperse template particles and one of the target particles (e.g., nucleic acids; paragraph [00028]). In the methodology of Abate, the target particles are sorted into droplets with the monodisperse template particles and droplet generation is accomplished such that about 70% to about 90% of droplets contain one, and not more than one (zero or one), monodisperse template particle and target nucleic acid (paragraphs [00081, 00146, and 00175]). 70% reads on about 10% given that the instant specification does not provide a limiting definition for the term “about”.
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Regan in view of Fei with that of Abate. One would be motivated to have about 10% of droplets contain zero or one target nucleic acid molecule given the assertion by Abate that this allows for accurate modeling for counting of amplification products in ddPCR (paragraph [00175]). One would have a reasonable expectation of success given that Abate indicates that ddPCR with droplets containing zero or one target molecule is a common methodology (paragraph [00175]).
Claims 49-50 are rejected under 35 U.S.C. 103 as being unpatentable over (Regan et al., WO 2015/023677 A1; cited on IDS of 8/27/2025) in view of Fei (Fei et al., WO 2020/001529 A1; EFD of June 7, 2018; cited on IDS of 4/4/2025) as applied to claims 1, 2, 6, 40-48, and 52 above, and further in view of Hindson (Hindson et al.; WO 2012/112970 A2; cited on PTO-892 of 10/01/2025).
The teachings of Regan in view of Fei are detailed above. Relevant to the instantly rejected claims, Regan in view of Fei teach amplifying a target nucleic acid in a discrete partition using a probe-antiprobe system to reduce background fluorescence and enable sensitive detection of amplified product.
Regan in view of Fei does not teach applying the probe-antiprobe (fluorescent probe-probe complement) methodology to chromosomal human DNA. However, use of qPCR with probe-antiprobe compositions to detect target nucleic acid molecules from human chromosomal DNA is known in the art, as taught by Hindson.
Hindson teaches amplifying a DNA locus in an emulsified droplet partition to produce a detectable signal and then detecting said signal (paragraph [0011]). Hindson teaches using fluorescer/quencher pairs in which fluorescence is increased as more amplification product is generated in amplification reactions (paragraphs [0125 and 0324-325]). Hindson teaches that the target can be human chromosomal DNA (paragraph [0082]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have used the method of Regan in view of Fei to study chromosomal loci of human DNA samples, as taught by Hindson. One would be motivated to do so given the assertion by Hindson that this methodology allows detection of human disease (such as trisomy 21) or rare allelic variants (such as BRAF V600E; paragraphs [0082] and [0444]). One would have a reasonable expectation of success given that Hindson successfully performs quantitative PCRs using detection probes in emulsified droplets on human chromosomal DNA (Examples 10-11 and 15-16).
Claims 51 and 53-54 are rejected under 35 U.S.C. 103 as being unpatentable over (Regan et al., WO 2015/023677 A1; cited on IDS of 8/27/2025) in view of Fei (Fei et al., WO 2020/001529 A1; EFD of June 7, 2018; cited on IDS of 4/4/2025) as applied to claims 1, 2, 6, 40-48, and 52 above, and further in view of Van Lode (Van Lode et al., WO 2010/130877 A2; cited on PTO-892 of 10/01/2025).
The teachings of Regan in view of Fei are detailed above. Relevant to the instantly rejected claims, Regan in view of Fei teach amplifying a target nucleic acid in a discrete partition using a probe-antiprobe system to reduce background fluorescence and enable sensitive detection of amplified product.
Regan in view of Fei does not explicitly teach that the probe complement comprises 15-25 nucleotides, that the melting temperature of the probe complement in relation to the fluorescent probe is between 40º C to 50º C, or that the melting temperature of the fluorescent probe in relation to the nucleic acid molecule is higher than the melting temperature of the complement probe in relation to the fluorescent probe (see the 112b rejection of claim 54 above).
However, usage of probe pairs which varying melting temperatures for detection of amplified nucleic acid targets is known in the art, as taught by Von Lode.
Von Lode teaches a competitive hybridization system in which a first probe with a fluorescent reporter (fluorescent probe) and a complementary probe (probe complement) with a quencher are used for detection of a target nucleic acid (Abstract and pg 8, ln 11-17). Von Lode teaches that the melting temperature (Tm) of the fluorescent reporter probe with the target nucleic acid is higher than the Tm of the fluorescent reporter probe with the probe complement (Abstract). Von Lode teaches a fluorescent probe that is between 16-22 nucleotides long and a probe complement that is between 16-22 nucleotides long (Table 1, pg 33). Von Lode teaches that the Tm of the fluorescent probe with probe complement is 46.1º C while the Tm of the fluorescent probe with the target nucleic acid is 55.7º C (which is higher than 46.1º C).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have used the method of Regan in view of Fei with that of Von Lode. One would be motivated to have differing melting temperatures between the complementary probes and the fluorescent probes and their target nucleic acid, given the assertion by Von Lode that this enables easy design and optimization of probe pairs for analysis of target regions without extensive experimental assay optimization (pg 20, ln 15-22) and with a higher Tm between the probes and their targets rather than each other ensures that the target nucleic acid competes for probe binding more strongly than the probes do for each other which leads to “increased sensitivity of detection” (pg 8, ln 26-29 and pg 9, ln 1-2). One would have a reasonable expectation of success given that Von Lode demonstrates successful detection of an amplified target nucleic acid using a fluorescent probe that, at higher temperatures, binds more strongly to the target than to a quencher probe (Example 1, pg 29-33).
Response to Remarks
Applicant's arguments filed 1/27/2026 have been fully considered but they are not persuasive for the following reasons.
Applicant traverses the rejection of claims 1-2, 6, 40-48 and 52 under 35 U.S.C. 103 over Regan in view of Fei (pg 10-12 of Remarks of 1/27/2026). While previous rejections described in the Office Action of 10/01/2025 have been withdrawn in light of Applicant’s amendments to the claims, rejections against 1-2, 6, 40-44, 46-48, and 52 are still being rejected under 35 USC 103 over Regan in view of Fei, and therefore Applicant’s arguments regarding the previous rejection shall be addressed. Applicant argues that “a skilled artisan would not have been motivated to modify the digital PCR methods of Regan with the 3D imaging technique of Fei for at least the reason that 1) combining Regan with Fei would frustrate the intended purpose of Regan and 2) they would not have a reasonable expectation of success” (pg 10 of Remarks). The examiner respectfully disagrees.
First, the Applicant is improperly characterizing the intended purpose of Regan. Applicant asserts that “Regan involves detecting droplets flowing through the detection module” and that the “purpose of Regan [is] to detect droplet flowing through a system”. However, the intended purpose of Regan is “performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another” wherein “[a] property of the target, such as concentration of the target, may be determined based on the photoluminescence detected” (Abstract). The assembly in Figure 5 of Regan that is cited by Applicant is merely an “exemplary system” (pg 10 of Remarks) for carrying out the amplification assay of Regan, and as such is not the only way in which the methodology of Regan may be achieved. Moreover, Regan teaches that the system may be composed of assemblies that are unconnected to other assemblies wherein the material is transported manually between devices (pg 22, ln 19-22). Therefore, the methodology of Regan does not require that droplets are flowing past a detector and would not be “frustrated” by the stationary imaging as taught by Fei (pg 12 of Remarks).
Second, given that the stationary detection method of Fei would not frustrate the “intended purpose” of Regan, one would have a reasonable expectation of success in combining the stationary imaging technique of Fei with that of Regan. One of skill in the art would be motivated to do so given the rationales provided above and would be able to readily adapt the emulsion technology of Regan with the imaging technology of Fei, especially given that Fei is also performing particle emulsions in which amplification reactions are taking place. Fei even points out the short-comings of flowing droplets past a detector in a microfluidic system (NOT that this is required by Regan) and provides reasons/adaptations for using 3D imaging in a stationary mode to overcome said shortcomings (paragraphs [0038-0039]).
For these reasons, the rejection of claims 1-2, 6, 40-44, 46-48, and 52 under 35 U.S.C. 103 over Regan in view of Fei is maintained (absent claim 45 due to the cancellation of this claim). Additionally, the rejections of claims 9 (Regan in view of Fei and further in view of Abate), 49-50 (Regan in view of Fei and further in view of Hindson), and 51 and 53-54 (Regan in view of Fei and further in view of Van Lode) are deemed proper and are maintained.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Anne Gussow can be reached at (571)272-6047. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683
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