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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/02/2025 has been entered.
Regarding the Office action mailed 09/04/2025, the rejections set forth therein are withdrawn in view of the amendments.
New grounds of rejection are set forth below.
Claim Rejections - 35 USC § 103
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.
Claim(s) 1-3, 5, 6, 8 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Kuhns (Blood Advances 3(2):136-147 (2019); IDS reference; cited by the European Patent Office) in view of Ney (Archives of Pathology & Laboratory Medicine 136(9):983-992 (2012); IDS reference; cited by the European Patent Office), Fiorini (US 2015/0151301) and Tanaka (US 2019/0352699).
Regarding claims 1-3 and 8, Kuhns disclosed:
A DNA detection method comprising the steps of: placing a DNA solution in each of a plurality of partitions
Fig. 1 and section entitled “Genotyping with ddPCR” beginning on page 140; “oil-enclosed reaction droplets”.
wherein the DNA solution may contain a plurality of types of DNAs including a first gene and a pseudogene of the first gene,
Fig. 1 and section entitled “Genotyping with ddPCR” beginning on page 140; NCF1 and pseudogenes NCF1B and NCF1C.
and the DNA solution contains a fluorescent-labeled probe or a DNA intercalator;
Fig. 1 and section entitled “Genotyping with ddPCR” beginning on page 140; two distinct probes: one giving blue fluorescence for the gene NCF1, the other giving green fluorescence for the pseudogenes NCF1B and NCF1C.
performing PCR in each of the partitions;
Fig. 1 and section entitled “Genotyping with ddPCR” beginning on page 140; “PCR reaction was performed on a BioRad T100 thermal cycler…”.
discriminating the type of DNA for each of the partitions…and counting the number of partitions for each type of the DNA;
Fig. 1 and section entitled “Genotyping with ddPCR” beginning on page 140; whether a droplet contained a copy of the gene (NCF1) or a pseudogene (NCF1B or NCF1C) was determined based on the distinguishable fluorescence signals of the corresponding probes.
outputting the number of partitions counted for each type of the DNA;
Fig. 1; “The droplets were analyzed to determine the number of “Blue” and “Green” nanodrops.” Section entitled “Genotyping with ddPCR” beginning on page 140; “…and the data were analyzed using BioRad QuantaSoft 1.5 to determine the number of blue GTGT and green ΔGT droplets.”
determining whether a ratio of the number of partitions in which the pseudogene is placed to the number of partitions in which the first gene is placed is a value within a range corresponding to 1, a value within a range corresponding to 1/2, a value within a range corresponding to 2, a value within a range corresponding to 0, or a value not within any range;
See Fig. 1, which indicates that a “normal” individual has 4 copies of ΔGT (two pseudogenes on each maternal and paternal chromosome 7) and 2 copies of GTGT (two normal NCF1 genes, one on each maternal and paternal chromosome 7). This is a ratio of pseudogene to gene of 4:2, or 2, for a normal individual.
Regarding claim 5, Kuhns displayed the “number” of first genes (Table 1, column headed “No. of GTGT droplets”).
Regarding claim 6, Kuhns disclosed that “the DNA has been diluted to minimize the number of double-colored droplets” (figure 1), which can reasonably be considered “limiting dilution”.
The difference between Kuhns and the claimed invention is: (1) Kuhns did not change a temperature of each of the partitions during the PCR or after the PCR and measuring a fluorescence intensity changing with the temperature change for each of the partitions;
calculate, for each of the partitions, a melting temperature of a double strand DNA placed in the partition, based on a change in the fluorescence intensity accompanying the temperature change; or
discriminate the type of DNA for each of the partitions based on the melting temperature;
(2) Kuhns did not explicitly disclose determining whether or not the ratio conforms to a predetermined value or range, and if the ratio conforms the predetermined value or range, displaying information indicating conformity, and if the ratio does not conform the predetermined value or range, displaying information indicating non-conformity; and
(3) Kuhns did not use probes for a wild-type and pseudogene modified with a same fluorescent element or a same probe for a wild-type and a pseudogene; Kuhns used different probes for the wild-type and pseudogenes, each labeled with a different fluorescent label.
(4) Kuhns did not determine whether the measured melting temperature was within a predetermined range.
Regarding the first difference, Ney disclosed the use of differences in melting temperature between an amplicon and a labeled probe to distinguish between an amplicon derived from a gene (KRAS) and its pseudogene (see figure 2, description of parts A and B). Regarding claim 9, as seen in Ney’s figure 2, the melting temperature corresponds to an inflection point (a peak) of the fluorescence intensity as temperature increases. Regarding the third difference, Ney used a same probe for the wild-type and pseudogene. Specifically, Ney used a pair of oligonucleotides, one labeled with fluorescein, the other labeled with LC Red 640, which constituted the probe (or, one could consider each oligonucleotide to be a “probe”). In any event, the same pair of oligonucleotides was designed to hybridize adjacently on both the wild-type and the pseudogene; see page 986, section entitled “Melting-Curve Analysis by Using Hybridization Probes” and Figure 2 description.
Fiorini disclosed a device for performing digital PCR (abstract) which could also be used to perform a melt curve analysis on each droplet following the PCR (paragraph [0081]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Kuhns by distinguishing between droplets containing a gene and droplets containing a pseudogene using melt curve analysis technique of Ney, including the use of the alternative probe format of Ney, whereby a same probe was used for both the wild-type gene and the pseudogenes, thereby arriving at the first and third differences between the claimed invention and Kuhns discussed above. It would have been obvious to do this because it represents an alternative probe format known in the prior art for distinguishing a gene from its pseudogene. MPEP 2143 discusses several rationales that may be used to support a conclusion of obviousness. One such rationale is the simple substitution of one known element for another to obtain predictable results. Another is choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success. Either of these rationales fits the scenario here. Kuhn showed that wild-type and pseudogene sequences could be distinguished using different probes labeled with different fluorescent labels, and Ney showed that wild-type and pseudogene sequences could be distinguished using a “same” probe for both, where distinction was made based on melting curve analysis.
Fiorini provided a device for performing melt curve analysis in the context of ddPCR. Therefore, one of ordinary skill in the art would have had a reasonable expectation of success in using Fiorini’s device to perform such modification of Kuhns’ method.
Regarding the second difference, Kuhns touted the method as a way to identify patients with and carriers of p47phox chronic granulomatous disease (CGD); see abstract. Therefore, it would have been obvious to use the assay to report whether someone was normal (i.e. had a pseudogene to gene of 4:2, or 2) (thus, conforming to that ratio) or not (thus, not conforming to that ratio).
Regarding the fourth difference, Tanaka taught determining whether a melting temperature was within a predetermined range corresponding to a specific target; see paragraphs [0012]-[0014] and claims 6-8.
It would have been prima facie obvious to determine whether a measured melting temperature was within a predetermined range corresponding to a particular target, thus identifying the target, since Tanaka disclosed this technique. Moreover, it would be obvious that the recited ratio of the claim could not be determined until the number of droplets containing the gene and pseudogene had been determined.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Kuhns (Blood Advances 3(2):136-147 (2019); IDS reference; cited by the European Patent Office) in view of Ney (Archives of Pathology & Laboratory Medicine 136(9):983-992 (2012); IDS reference; cited by the European Patent Office) and Fiorini (US 2015/0151301) and Tanaka (US 2019/0352699) as applied to claims 1-3, 5, 6, 8 and 9 above, and further in view of Yancy (US 2012/0088236).
The disclosures of Kuhns, Ney and Fiorini have been discussed. None of these references disclosed determining the type of DNA in each partition based on information indicating a predetermined reference melting temperature.
Yancy disclosed (paragraph [0045]): “… following amplification, high resolution melt analysis is used to determine the melting temperature (or melting point) of the amplified genetic material. The melting temperature (as shown by a derivative plot of -(d/dT) fluorescence vs. temperature), can then be compared to the melting temperature of a known sample (wildtype, homozygous or heterozygous) in order to determine whether a mutation or other variant is present.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined disclosures of Kuhns, Ney and Fiorini by comparing the melting temperature observed for a partition to known (i.e. predetermined) melting temperatures for expected products (either the NCF1 gene or the NCF1B or NCF1C pseudogenes), i.e. “reference melting temperatures” in order to determine the species present in the partition, since comparing observed melting temperatures to known melting temperatures for expected species in order to determine which species was present in a sample was old and well-known, as demonstrated by Yancy.
Claims 10 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Kuhns (Blood Advances 3(2):136-147 (2019); IDS reference; cited by the European Patent Office) in view of Ney (Archives of Pathology & Laboratory Medicine 136(9):983-992 (2012); IDS reference; cited by the European Patent Office) and Fiorini (US 2015/0151301) and Tanaka (US 2019/0352699) as applied to claims 1-3, 5, 6, 8 and 9 above, and further in view of Frayley (US 2021/0241857).
The disclosures of Kuhns, Ney and Fiorini have been discussed. Regarding the device Fiorini disclosed for performing droplet digital PCR and subsequent melting curve analysis, Fiorini disclosed an optical detector which could be an “image sensor” (paragraph [0063]): “For example, the detector 108 may be an image sensor located underneath the complete first micro-fluidic channel 104 wherein the size of the detector 108 allows monitoring of droplets during propagation through the complete first micro-fluidic channel 104.” Note that Fiorini’s device was also a device capable of arranging a DNA solution in each of a plurality of partitions (i.e. micro-fluidic channel 104, which arranges the droplets created by droplet generator 107 within the channel; paragraph [0044], [0045] and Fig. 1).
Fiorini’s device also comprised a temperature adjustment unit that changed a temperature of each droplet in order to perform PCR (paragraph [0044] and Fig. 1: heating element 101 thermally coupled to the first micro-fluidic channel 104; the heating element 101 is a single heating element connected to a temperature control unit 111 configured to cycle the temperature of the complete first micro-fluidic channel 104 through at least two temperature values).
Fiorini also disclosed the device could capture an image in order to acquire a fluorescence intensity changing with a temperature change for each of the partitions; paragraph [0063]: “…wherein the size of the detector 108 allows monitoring of droplets during propagation through the complete first micro-fluidic channel 104”; paragraph [0081]: “…a heating element is located at the outlet 103 of the first micro-fluidic channel 104. The heating element may be configured to continuously heat droplets to increasing temperatures. The temperature of the heating element can be increased for a certain period of time, e.g. increasing the temperature of the heating element for 10 minutes. The droplets are exposed to continuously increasing temperatures, e.g. an increasing temperature from 60 to 90 degrees Celsius within 10 minutes. By continuously increasing the temperature to heat the droplets at the outlet, a melting curve analysis of PCR products can be performed.”
Fiorini also disclosed (paragraph [0064]): “According to an embodiment of the disclosure, the micro-fluidic device 100 further comprises a computing unit 110 connected to the detector 108. The computing unit 110 is configured for determining a percentage of droplets containing PCR products.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to carry out the method of Kuhn, modified to discriminate based on melting curve analysis as per the disclosure of Ney, using the device of Fiorini for the reasons discussed for claim 1. Therefore, it would also have been obvious to modify the computing unit to carry out the analytical process of counting droplets containing the NCF1 gene and the NCF1B and NCF1C pseudogenes, and to display the data as per the method of Kuhn to report the sample as being normal, or being affected by or a carrier of p47phosx CGD.
None of Kuhns, Ney or Fiorini mentioned a database that stored reference melting temperatures for the NCF1 gene and the NCF1B and NCF1C pseudogene amplicons or a memory storing melting temperatures detected for each of the partitions (though this would have been obvious in order for the computing unit of Fiorini’s device to carry out the method of Kuhns whereby the number of droplets containing NCF1 gene and the NCF1B and NCF1C pseudogenes was determined; i.e. a memory to hold the data would have been necessary for this purpose).
Frayley disclosed matching a test melt curve to a melt curve contained in a pre-defined database (paragraph [0096], [0102]). Frayley also disclosed (paragraph [0090]): “The analysis device processor can be configured to exclude melt curves below a threshold melt temperature to profile the sequence of a target nucleic acid. In some embodiments, exclusion of melt curves below a threshold melt temperature can improve type I and II errors by correcting for short non-specific amplification products that melt at a lower temperature than the target amplicon.”
It would have been prima facie obvious to one of ordinary skill in the art to determine the species present in each droplet by comparing the obtained melting temperature to a database of reference temperatures as disclosed by Frayley to properly identify which species was present in each droplet. It would also have been obvious to perform thresholding as disclosed by Frayley in order to “improve type I and II errors by correcting for short non-specific amplification products that melt at a lower temperature than the target amplicon”.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1-3, 5-10 and 12 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
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/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681