DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, drawn to a method of quantifying a target nucleic acid analyte by performing amplification (claims 1-25) in the reply filed on 06/07/2024 was acknowledged in the office action mailed 02/25/2025.
Applicant Response
Applicant's response, filed 12/16/2025, has been fully considered. Rejections and/or objections not reiterated from previous Office Actions are hereby withdrawn. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Claim Status
Claims 3, 23 and 26-44 are cancelled.
Claims 1-2, 4-22 and 24-25 are pending and under examination herein.
Claims 1-2, 4-22 and 24-25 are rejected.
Priority
The instant application is the National Stage entry of PCT/US2019/041260 , International Filing Date: 07/10/2019, which claims priority from Provisional Application 62/764946 , filed 08/17/2018 and Provisional Application 62/696147 , filed 07/10/2018. As such, the effective filing date assigned to each of claims 1-2, 4-22 and 24-25 is 07/10/2018.
Drawings
The drawings were accepted by the examiner in the office action mailed 10/22/22025.
Claim Rejections - 35 USC § 112
The rejection of claim 3 under 35 U.S.C. 112(d) is withdrawn in view of cancelation of the claim in the claim amendments filed 12/16/2025.
Claim Rejections - 35 USC § 101
The rejection of claims 3 and 23 under 35 U.S.C. 101 is withdrawn in view of cancelation of the claim in the claim amendments filed 12/16/2025.
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-2, 4-22 and 24-25 remain rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea/law of nature/natural phenomenon without significantly more. In accordance with MPEP § 2106, claims found to recite statutory subject matter (Step 1: YES) are then analyzed to determine if the claims recite any concepts that equate to an abstract idea, law of nature or natural phenomenon (Step 2A, Prong 1). Newly recited portions are necessitated by claim amendments.
In the instant application, the claims recite the following limitations that equate to an abstract idea and a law of nature or natural phenomenon:
Claims 1 and 11 recites (c1) determining a slope of the baseline segment between the starting cycle and the baseline end-cycle; (c2) for each of a plurality of cycles or times at which a fluorescence measurement was obtained after the baseline end-cycle, adjusting the fluorescence measurement by subtracting a fixed adjustment value dependent on the slope of the baseline segment and the baseline end- cycle, wherein the fixed adjustment value is the product of multiplying the slope of the baseline segment by the reaction cycle number of the baseline end-cycle; and (d) determining a cycle threshold (Ct) value from values comprising at least a portion of the adjusted fluorescence measurements from step (c2), or determining that the target nucleic acid analyte is absent or not present in an amount above a limit of detection, thereby quantifying the target nucleic acid analyte.
Claim 2 recites wherein the fixed adjustment value is less than the product of multiplying the slope of the baseline segment by reaction cycle numbers greater than the cycle number of the baseline end-cycle.
Claim 3 recites wherein the fixed adjustment value is the product of multiplying the slope of the baseline segment by the reaction cycle number of the baseline end-cycle.
Claim 4 recites further comprising, after step (b) and before step (c), the step of smoothing at least a portion of the fluorescence measurements.
Claim 5 recites wherein smoothing comprises applying a moving average to the portion of the fluorescence measurements.
Claim 6 recites wherein applying the moving average comprises averaging across M cycles, wherein M is 3, 4, 5, 6, 7, 8, 9, 10, or 11.
Claim 7 recites wherein smoothing at least a portion of the fluorescence measurements comprises either polynomial curve fitting or spline smoothing.
Claim 8 recites further comprising leveling fluorescence measurements so that no fluorescence measurement has a value less than zero.
Claim 9 recites further comprising performing crosstalk correction on fluorescence measurements from the first fluorophore of the first detection probe.
Claim 10 recites wherein crosstalk correction comprises subtracting an estimate of bleed-through signal from a second fluorophore of a second detection probe from a fluorescence signal measured for the first fluorophore, wherein the second detection probe comprises the second fluorophore, wherein the second fluorophore and the first fluorophore have overlapping emission spectra, and wherein the estimate of bleed-through signal is dependent on contemporaneous fluorescence measurements from the second fluorophore and a predetermined ratio of observed fluorescence from the second fluorophore to expected bleed-through signal from the second fluorophore in the fluorescence measurements of the first fluorophore.
Claim 11 further recites for each of a plurality of cycles or times at which a fluorescence measurement was obtained for the baseline segment, adjusting the fluorescence measurement by subtracting a variable adjustment value dependent on the slope of the baseline segment and the cycle or time at which the measurement was obtained.
Claim 12 recites further comprising a conversion region exclusion step, wherein a user-defined number of cycles following initiation of the cycled amplification reaction are eliminated, thereby identifying the starting cycle of the baseline segment as the next remaining cycle number.
Claim 13 recites further comprising a baseline end-cycle identification step that comprises calculating slopes between fluorescence measurements for adjacent pairs of cycles in the cycled amplification reaction, and determining when a predetermined slope is reached, thereby identifying the baseline end-cycle.
Claim 14 recites further comprising a baseline end-cycle identification step that comprises calculating slopes between fluorescence measurements at adjacent pairs of cycles in the cycled amplification reaction, and determining when a predetermined percentage increase is reached, thereby identifying the baseline end-cycle.
Claim 17 recites wherein step (e) comprises: (i) subtracting a minimum value of the adjusted fluorescence measurements of step (d) from the maximum value of the adjusted fluorescence measurements of step (d), thereby providing a fluorescence range value; and (ii) determining that the target nucleic acid analyte is not present in an amount equal to or greater than a predetermined limit of detection if the fluorescence range value is less than or equal to a predetermined threshold.
Claim 18 recites wherein at least one adjusted fluorescence measurement after the baseline end-cycle is greater than or equal to a predetermined threshold, and wherein the Ct value is determined in step (d) as the earliest cycle number at which the adjusted fluorescence measurement is greater than or equal to the predetermined threshold.
Claim 19 recites wherein at least one adjusted fluorescence measurement from step (d) is greater than or equal to a predetermined threshold, and wherein the Ct value is determined from values comprising: (i) the cycle in which the earliest adjusted fluorescence measurement greater than or equal to the predetermined threshold occurred; (ii) the earliest adjusted fluorescence measurement greater than or equal to the predetermined threshold; or (iii) a value of an adjusted fluorescence measurement from a cycle preceding the cycle in which the earliest adjusted fluorescence measurement greater than or equal to the predetermined threshold occurred.
Claim 20 recites wherein the Ct value is estimated from an interpolation of fluorescence values between adjusted fluorescence measurements from the cycle in which the earliest adjusted fluorescence measurement greater than or equal to the predetermined threshold occurred and the preceding cycle.
Claim 21 recites wherein the interpolation is a linear interpolation.
Claim 22 wherein the CT value is a fractional cycle value corresponding to the predetermined threshold in the interpolation.
Claim 23 recites smooth at least a portion of the fluorescence measurements, determining the slope of the baseline segment, adjust the fluorescence measurements, and determine the Ct value or that the target nucleic acid analyte is absent or not present in an amount above a limit of detection.
These recitations equate to steps of collecting information, analyzing data and making observations, evaluations and judgements that can be carried out in the human mind. Specifically, obtaining fluorescence measurements during a plurality of cycles of the cycled amplification reactions, determining a slope of the baseline segment, adjusting the fluorescence measurements for the cycles, determining a Ct values, or determining whether the target nucleic acid analyte is absent or present, smoothing or leveling the measurements, performing crosstalk correction, a conversion region exclusion step to identify the starting cycle of the baseline segment as the next remaining cycle number, and the baseline end-cycle identification steps can be practically performing the human mind as claimed and are similar to the concepts of collecting and comparing known information in Classen Immunotherapies, Inc. v. Biogen IDEC, 659 F.3d 1057, 1067, 100 USPQ2d 1492, 1500 (Fed. Cir. 2011) and collecting information, analyzing it, and reporting certain results of the collection and analysis in Electric Power Group v. Alstom, S.A., 830 F.3d 1350, 1353-54, 119 USPQ2d 1739, 1741-42 (Fed. Cir. 2016) that the courts have identified as concepts that can be practically performed in the human mind. Therefore, each of the above recited limitations fall under the “Mental Processes” grouping of abstract ideas. Furthermore, the steps as claimed for determining a slope of the baseline segment, adjusting the fluorescence measurements for the cycles, determining a Ct values, smoothing or leveling the measurements, and performing crosstalk correction equate to organizing information and manipulating information through mathematical correlations and reciting a mathematical equation, similar to the concepts of taking existing information, manipulating the data using mathematical functions, and organizing this information into a new form in Digitech Image Techs., LLC v. Electronics for Imaging, Inc., 758 F.3d 1344, 1350, 111 USPQ2d 1717, 1721 (Fed. Cir. 2014). Therefore, these limitations fall under the “Mathematical Concepts” grouping of abstract ideas. Claims 2, 5-7 and 17-22 further qualify the recited judicial exceptions. As such, claims 1-2, 4-22 and 24-25 recite an abstract idea (Step 2A, Prong 1: YES).
Claims found to recite a judicial exception under Step 2A, Prong 1 are then further analyzed to determine if the claims as a whole integrate the recited judicial exception into a practical application or not (Step 2A, Prong 2). The judicial exceptions are not integrated into a practical application because the claims do not recite an additional element that reflects an improvement to technology or applies or uses the recited judicial exception to affect a particular treatment for a condition. Rather, the instant claims recite additional elements that amount to mere instructions to implement the abstract ideas in a generic computing environment and merely indicating a field of use or technological environment in which to apply a judicial exception. Specifically, the claims recite the following additional elements:
Claims 1 and 11 recite wherein the method is performed using a nucleic acid analyzer that includes a thermocycler configured to regulate the temperature of the sample, at least one fluorometer configured to measure fluorescence from the sample, and a processor and a memory operably linked to the fluorometer and the thermocycler and storing instructions to be executed by the processor to perform steps of the method, (a) performing, with the thermocycler in communication with the processor, a cycled amplification reaction on the sample in the presence of a first detection probe labeled with a first fluorophore, wherein the first fluorophore exhibits target nucleic acid analyte-dependent fluorescence; (b) obtaining, with the at least one fluorometer in communication with the processor, fluorescence measurements during a plurality of cycles of the cycled amplification reaction, and storing the fluorescence measurements in the memory, wherein a plurality of the obtained fluorescence measurements constitute a baseline segment that begins at a starting cycle, and terminates at a baseline end-cycle that precedes detectable amplification of the target nucleic acid analyte .
Claim 15 recites wherein the first detection probe further comprises a quencher moiety in energy transfer relationship with the first fluorophore.
Claim 16 recites wherein the first detection probe further comprises a quencher or a FRET acceptor, and either: (i) comprises a self-complementary region and undergoes a conformational change upon hybridization to the target nucleic acid analyte that reduces quenching of or FRET transfer from the first fluorophore; or (ii) undergoes exonucleolysis following hybridization to the target nucleic acid analyte that releases the first fluorophore from the first detection probe, thereby resulting in increased fluorescence; or (iii) undergoes cleavage following hybridization to a fragment of a primary probe that was cleaved following hybridization to the target nucleic acid analyte, and cleavage of the first detection probe releases the first fluorophore, thereby resulting in increased fluorescence.
Claim 24 recites wherein the at least one fluorometer is configured to detect fluorescence in a plurality of channels.
Claim 25 recites wherein the cycled amplification reaction is a polymerase chain reaction.
These claims recite limitations for gathering data. The amplification reaction steps and obtaining fluorescence measurements steps are performed to gather data to perform the mental steps and mathematical processes, and the system components of the fluorometer and a thermocycler apparatus are equipment to collect data to perform the mental steps and mathematical processes, and there is no indication that these steps are impacted by the recited the judicial exception, and therefore the recited judicial exception is not integrated into these additional elements. These limitations equate to mere data gathering, which the courts have found to be insignificant extra-solution activity (see MPEP 2106.05(g)).
Furthermore, the claims also recites using a generic computing systems and computer program products to carry out instructions to implement an abstract idea on a computer. The use of a computer or other machinery in its ordinary capacity for economic or other tasks (e.g., to receive, store, or transmit data) or simply adding a general purpose computer or computer components after the fact to an abstract idea (e.g., a fundamental economic practice or mathematical equation) does not integrate a judicial exception into a practical application. See Affinity Labs v. DirecTV, 838 F.3d 1253, 1262, 120 USPQ2d 1201, 1207 (Fed. Cir. 2016) (cellular telephone); TLI Communications LLC v. AV Auto, LLC, 823 F.3d 607, 613, 118 USPQ2d 1744, 1748 (Fed. Cir. 2016) (computer server and telephone unit).
Therefore, there is no indication that any of these additional elements provide a practical application of the recited judicial exception outside of the judicial exception itself. As such, claims 1-2, 4-22 and 24-25 are directed to an abstract idea (Step 2A, Prong 2: NO).
Claims found to be directed to a judicial exception are then further evaluated to determine if the claims recite an inventive concept that provides significantly more than the judicial exception itself (Step 2B). Further analyzing the additional elements under step 2B, the additional elements as described above do not rise to the level of significantly more than the judicial exception. As directed in the Berkheimer memorandum of 19 April 2018 and set forth in the MPEP, determinations of whether or not additional elements (or a combination of additional elements) may provide significantly more and/or an inventive concept rests in whether or not the additional elements (or combination of elements) represents well-understood, routine, conventional activity. Said assessment is made by a factual determination stemming from a conclusion that an element (or combination of elements) is widely prevalent or in common use in the relevant industry, which is determined by either a citation to an express statement in the specification or to a statement made by an applicant during prosecution that demonstrates a well-understood, routine or conventional nature of the additional element(s); a citation to one or more of the court decisions as discussed in MPEP 2106(d)(II) as noting the well-understood, routine, conventional nature of the additional element(s); a citation to a publication that demonstrates the well-understood, routine, conventional nature of the additional element(s); and/or a statement that the examiner is taking official notice with respect to the well-understood, routine, conventional nature of the additional element(s).
With respect to the instant claims under the 2B analysis, the limitations of performing a cycled amplification reaction on nucleic acid samples is well-understood, routine, conventional activity (see Genetic Techs. Ltd. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed. Cir. 2015); University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014). Furthermore, the prior art to Lo et al. (Clinical Applications of PCR 2006, Vol 16, Springer Science and Business Media; previously cited) discloses performing PCR with fluorescent primers, including using traditional hybridization probe design with an acceptor and donor fluorophore, using systems that can automate PCR with optical thermocyclers that regulate temperature and fluorometers that detect fluorescent probes is well-known in the art (Chapter 1, sections 10, 11.3, 11.5 -11.6; chapter 2, section 1-3; chap 3, section 3.5, fig 5). The instant specification also discloses such nucleic acid analyzers are commercially available (para 00176).
Furthermore, the use of a computer or other machinery in its ordinary capacity for economic or other tasks (e.g., to receive, store, or transmit data) or simply adding a general purpose computer or computer components after the fact to an abstract idea (e.g., a fundamental economic practice or mathematical equation) does not provide significantly more. See Affinity Labs v. DirecTV, 838 F.3d 1253, 1262, 120 USPQ2d 1201, 1207 (Fed. Cir. 2016) (cellular telephone); TLI Communications LLC v. AV Auto, LLC, 823 F.3d 607, 613, 118 USPQ2d 1744, 1748 (Fed. Cir. 2016) (computer server and telephone unit).
Therefore, the additional elements do not comprise an inventive concept when considered individually or as an ordered combination that transforms the claimed judicial exception into a patent-eligible application of the judicial exception, and the claims do not amount to significantly more than the judicial exception itself (Step 2B: NO). As such, claims 1-2, 4-22 and 24-25 are not patent eligible.
Response to applicant’s arguments
Applicant asserts the claims recites steps that cannot be carried out in the human mind, such as steps for generating and obtaining fluorescent measurements, and computation steps performed with a processor and memory of the nucleic acid analyzer, and further asserts the claimed method improves the functioning of a nucleic acid analyzer through the improvement in the curve appearance, and request withdrawal of the rejection (Applicant’s Arguments, p 9, para 5-p 11, para 3).
It is respectfully submitted that this is not persuasive. While the examiner agrees the physical steps for generating and obtaining data do not recite mental process, the claims still recite judicial exceptions as discussed above. Furthermore, under Step 2A, prong 2, the improvement appear to be to and provided solely by the analysis steps themselves; the nucleic acid analyzer itself does not seem affected by the recited judicial exceptions. As discussed in MPEP 2106.05(a), the judicial exception alone cannot provide the improvement and the improvement cannot be to the judicial exception itself. Therefore, there is no indication that any of these additional elements provide a practical application of the recited judicial exception outside of the judicial exception itself and claims 1-2, 4-22 and 24-25 are directed to an abstract idea. Therefore, the rejection is maintained.
Claim Rejections - 35 USC § 102
The rejection of claims 3 under 35 U.S.C. 102(a)(1) as being anticipated by Li (US20130273547A1; 10/05/2023 IDS; previously cited) is withdrawn in view of cancelation of the claim in the claim amendments filed 12/16/2025.
The rejection of claims 1, 4-8, 11-22 and 25 under 35 U.S.C. 102(a)(1) as being anticipated by Li (US20130273547A1; 10/05/2023 IDS; previously cited) is withdrawn in view of claim amendments filed 12/16/2025, as Li does not discloses the method is performed using a nucleic acid analyzer that includes a thermocycler configured to regulate the temperature of the sample and at least one fluorometer.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 4-8, 11-22 and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Li (US20130273547A1; 10/05/2023 IDS; previously cited) and further in view of Lo et al. (Clinical Applications of PCR 2006, Vol 16, Springer Science and Business Media; previously cited; hereafter referred to as Lo) and Buse et al. (US 2016/0032358; newly cited; hereafter referred to as Buse) as evidenced by Heinz et al. (US20120221252A1; newly cited; hereafter referred to as Heinz).
With respect to claim 1 and 11, Li discloses methods correct for high-sloped baseline observed in real-time PCR curves and provides a way of verifying amplification efficiency using a statistical approach (abstract).
With respect to (a), Li discloses detecting an optical signal emitted during a real-time PCR amplification of a target nucleic acid, using fluorescent labels (claim 1; para 0100).
With respect to (b), Li discloses plotting the intensity of the optical signal as a function of cycle number to obtain a first real-time PCR plot having a baseline phase and an exponential phase and distinguishing the end cycle of the baseline phase from the starting cycle of the exponential phase (claim 1).
With respect to (c1), Li discloses generating a baseline slope fitting curve of the first plot, wherein the baseline slope fitting curve is a linear regression (claim 6).
With respect to (c2), Li discloses rotating the PCR curve counter-clockwise based on the angle of the baseline fitting curve, in which the angle is determined based on the slope of the baseline fitting curve, and which results in the subtraction of the total rise of the baseline (at the baseline end cycle) from all measurements, in order to flatten the baseline(i.e. the fixed adjustment value is dependent on the slope of the baseline segment and the baseline end-cycle) (para 0131-0134; para 0163-0164). Li discloses the rotation of the measurements using the slope is performed by rotating the measurements counter-clockwise to produce a flat baseline (indicating the slope is a positive), which removes the complete rise (i.e. slope multiplied by the baseline end cycle) of the baseline from all measurements (para 0131-0134; para 0163-0164). For further clarification, the rotation by the fixed adjustment value is applied to the entire curve, not just up to the baseline end-cycle, and therefore Li discloses “for each of a plurality of cycles or times at which a fluorescence measurement was obtained after the baseline end-cycle, adjusting the fluorescence measurement by subtracting a fixed adjustment value dependent on the slope of the baseline segment and the baseline end-cycle” (fig 4). Furthermore, as the slope=Δy/Δx, and the Δy (i.e the rise) is equal to slope multiplied by the Δx (i.e. baseline end-cycle number). Therefore, the rotation which flattens the baseline based on the slope of the baseline and removes the complete rise of the baseline teaches the limitation of “wherein the fixed adjustment value is the product of multiplying the slope of the baseline segment by the reaction cycle number of the baseline end-cycle”.
With respect to claim 11, step (c3), Li further discloses producing a third plot with a flat baseline by converting each fluorescent data point (x, y), in which x is the cycle number and y is fluorescence, into a modified data point (x’, y’) applying the formula,
x
'
y
'
=
c
o
s
θ
-
s
i
n
θ
s
i
n
θ
c
o
s
θ
x
y
,
in which
θ
is the angle corresponding to the slope of the base fitting curve (i.e. the slope converted to an angle) (para 0016-0017; fig 4-5; fig 8a).
With respect to claim 1, step (d) and claim 11, step (d), Li discloses in the traditional cycle threshold (Ct) method, the cycle number is determined based on the point within the exponential phase of the PCR curve where the fluorescence response increases above the baseline level to cross a predetermined fluorescence threshold value and further discloses calculating the crossing point of a sample as the point where the sample's fluorescence curve turns sharply upward, corresponding to the maximum of the second derivative of the amplification curve, using the corrected values (para 0003). Li further discloses determining the concentration of the target nucleic acid in the sample (para 0013).
With respect to claim 2, Li discloses the rotation of the measurements using the slope is performed by rotating the measurements counter-clockwise to produce a flat baseline (indicating the slope is a positive), which removes the complete rise (i.e. slope multiplied by the baseline end cycle) of the baseline from all measurements, and since the rotation only considers the total rise of the baseline fitting curve (i.e. slope and baseline end cycle), this value would be lower than the product of the slope and any cycle number greater than the baseline end cycle number (para 0131-0134; para 0163-0164).
With respect to claims 4-8, Li discloses rescaling the raw PCR data and using linear regression to determine a baseline fitting line (i.e. a polynomial function), which is then used to determine the slope (i.e. smoothing at least a portion of the fluorescence measurements between steps (b) and (c)) (para 0161; fig 4). Li further discloses plotting a 3-point moving Coefficient of Variance (CV) via polynomial equation, and distinguishing the ground phase and the exponential phase by CV of every 3 consecutive data points, in which the CV is determinized using the standard deviation, to determine the ground phase, in order to perform the linear regression (fig 8A; para 0014; para 0157-0161). While Li does not explicitly show the leveling of fluorescence values, Li discloses the sloped baseline of a real-time PCR curve is first converted to a curve with a flat baseline having near zero fluorescence, with the flat baseline having a positive slope, implying that all fluorescence values are above zero (para 0005; fig 11-12; para 141-142).
With respect to claim 12, Li discloses multiple ways for setting or determining the starting cycle, including that the starting cycle can be any specified cycle number (para 0014).
With respect to claims 13 and 14, Li discloses “The end cycle of the baseline phase or the starting cycle of the exponential phase is assigned as one of the followings: 1) a first cycle which shows a CV significantly greater than that of its previous cycle, and 2) a cycle having a CV that is equal to or greater than a specified value, e.g., 0.5. If no cycle satisfies the criteria in 1) and 2), the end cycle of the baseline phase (or the starting cycle of the exponential phase) is assigned as the total PCR cycles” (para 0014).
With respect to claims 15 and 16, Li discloses using probes designed so that donor emission is quenched in the absence of target by fluorescence resonance energy transfer (FRET) between two chromophores (para 105; fig 1-2).
With respect to claim 17, step (i), Li discloses further adjusting the PCR curve by minimizing the baseline fluorescence by subtracting the average baseline value of the flat baseline (i.e. minimum value) from the adjusted fluorescence values, which includes the maximum fluorescent value, thereby providing a fluorescence range in the exponential phase for analyte detection (para 0018; para 0165-0166; fig 6). It is noted that step (ii) is not required to be performed, as under the broadest reasonable interpretation, it is only required if the fluorescence range is less than or equal to a predetermined threshold.
With respect to claims 18-19, Li discloses in the traditional cycle threshold (Ct) method the cycle number is determined based on the point within the exponential phase of the PCR curve where the fluorescence response increases above the baseline level to cross a predetermined fluorescence threshold value (para 0003).
With respect to claims 20-22, Li discloses determining a S-shape curve function approximation of the real-time PCR curve of FIG. 11 and the tangent line at the inflection point (i.e. Ct value) (claim 20-21;para 0062-63; fig 14-15; para 148-150).
With respect to claim 25, Li discloses performing PCR (claim 11).
However, with respect to claims 1 and 11, Li does not discloses the using a thermocycler or fluorometer, and with respect to claim 24, that the fluorometer is configured to detect fluorescence in a plurality of channels.
However, the prior art to Lo, in the same field of endeavor, discloses performing PCR with fluorescent primers, including using traditional hybridization probe design with an acceptor and donor fluorophore, using systems that can automate PCR with optical thermocyclers that regulate temperature and fluorometers that detect fluorescent probes is well-known in the art and commercially available (Chapter 1, sections 10, 11.3, 11.5 -11.6; chapter 2, section 1-3; chap 3, section 3.5, fig 5).
Furthermore, the prior art to Buse, further discloses improved thermocycling of low volume nucleic acid amplification reactions, with an analyzer with a fluorometer, as described in Heinz, integrated with a thermocycler (para 0115-para 0141-0145; para 0293). Heinz discloses the fluorometer can detect fluorescence in a plurality of channels (para 014-0126).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to have used a nucleic acid analyzer with a thermocycler and a fluorometer to generate and obtain the data used in the analysis of Li, as this was a known technique at the effective filing date of the claimed invention.
Therefore, the invention is prima facie obvious.
Response to applicant’s arguments
Applicant states that Li does not disclose the limitations of “for each of a plurality of cycles or times at which a fluorescence measurement was obtained after the baseline end-cycle, adjusting the fluorescence measurement by subtracting a fixed adjustment value dependent on the slope of the baseline segment and the baseline end-cycle, wherein the fixed adjustment value is the product of multiplying the slope of the baseline segment by the reaction cycle number of the baseline end-cycle”, as Li discloses a two-step adjustment process with a second step of subtracting by the average fluorescent values of the baseline, and requests withdrawal of the rejections (Applicant’s Arguments, p 12, para 1-p 14, para 2).
It is respectfully submitted that this is not persuasive. The instant claims do not limit the adjustment steps to only the steps recited. Therefore, even if Li performs further adjustment steps, Li discloses the adjustment steps of the instant claims as well. As discussed above, Li discloses rotating the PCR curve counter-clockwise based on the angle of the baseline fitting curve, in which the angle is determined based on the slope of the baseline fitting curve, and which results in the subtraction of the total rise of the baseline (at the baseline end cycle) from all measurements, in order to flatten the baseline(i.e. the fixed adjustment value is dependent on the slope of the baseline segment and the baseline end-cycle) (para 0131-0134; para 0163-0164). Li discloses the rotation of the measurements using the slope is performed by rotating the measurements counter-clockwise to produce a flat baseline (indicating the slope is a positive), which removes the complete rise (i.e. slope multiplied by the baseline end cycle) of the baseline from all measurements (para 0131-0134; para 0163-0164). For further clarification, the rotation by the fixed adjustment value is applied to the entire curve, not just up to the baseline end-cycle, and therefore Li discloses “for each of a plurality of cycles or times at which a fluorescence measurement was obtained after the baseline end-cycle, adjusting the fluorescence measurement by subtracting a fixed adjustment value dependent on the slope of the baseline segment and the baseline end-cycle” (fig 4). Furthermore, as the slope=Δy/Δx, and the Δy (i.e the rise) is equal to slope multiplied by the Δx (i.e.baseline end-cycle number). Therefore, the rotation which flattens the baseline based on the slope of the baseline and removes the complete rise of the baseline teaches the limitation of “wherein the fixed adjustment value is the product of multiplying the slope of the baseline segment by the reaction cycle number of the baseline end-cycle”. Therefore, Li teaches the limitations of the instant claims. New art has been applied to teach the limitations of the thermocycler and the fluorometer.
Conclusion
No claims allowed.
Claims 9-10 appear to be free from prior art, as the closest prior art to Li (US20130273547A1; 10/05/2023 IDS; previously cited) does not appear to teach or suggest performing crosstalk correction on the fluorescence measurements.
Inquiries
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIDHI DHARITHREESAN whose telephone number is (571)272-5486. The examiner can normally be reached Monday - Friday 9:00 - 5:00.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Larry D Riggs II can be reached on (571) 270-3062. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/N.D./ Examiner, Art Unit 1686
/Karlheinz R. Skowronek/Supervisory Patent Examiner, Art Unit 1687