DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Response to Amendment
The amendment and terminal disclaimer filed 08/28/2025 overcome the claim objections and rejections set forth in the Office action mailed 08/25/2025. Upon closer review, new grounds of rejection are set forth below. This Office action is NON-FINAL.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 pre-AIA 35 U.S.C. 103(a) 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 2-8, 11-13, 19 and 24-26 is/are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable Beer (Anal. Chem. 79:8471-8475 (2007), IDS ref) over Blow (Nature Methods 4(10)869-875 (2007)).
Regarding claim 2, Beer disclosed:
A method of quantifying a target nucleic acid of a sample, the method comprising: dividing the sample into a plurality of subvolumes, said plurality of subvolumes comprising at least a first set of subvolumes each of which comprises one or more target nucleic acid, and a second set of subvolumes which do not contain the target nucleic acid;
Page 8472, paragraph spanning columns: “Here we report on the first on-chip digital microfluidic real-time PCR instrument for generating monodisperse microdroplet reactors, thermal cycling them for PCR, and detecting real-time amplification in the individual picoliter droplets.”
Page 8473, first paragraph under “Results And Discussion”: “With this droplet generation design, the aqueous sample stream encounters the viscous mineral oil cross-flow at the T-junction creating a shear zone. When shear stress overcomes surface tension, the extended aqueous bolus breaks off and quickly relaxes to a spherical geometry as it passes downstream.”
See Table 3, which shows different experiments with different target concentrations. Note that experiments were run with an average of 0.4 or 0.06 copies of genomic DNA per droplet. This would necessarily mean that some droplets contained no copies of genomic DNA.
See Figure 3b, which shows the results of amplification with an average of 0.06 copies per droplet, noting that only two droplets amplified; see last paragraph on page 8474: “Examining Figure 3b and adjusting the droplet count to include three doublets and two each of the 3× and 4× droplets gives an equivalent total of 42 droplets, 2 of which showed obvious amplification…”.
subjecting the plurality of subvolumes to an amplification reaction during which the target nucleic acid is amplified;
Page 8472, paragraph spanning columns: “Here we report on the first on-chip digital microfluidic real-time PCR instrument for generating monodisperse microdroplet reactors, thermal cycling them for PCR, and detecting real-time amplification in the individual picoliter droplets.”
See Figure 3b, which shows the results of amplification with an average of 0.06 copies per droplet.
monitoring amplification of the target nucleic acid during the amplification reaction;
Page 8472, paragraph spanning columns: “Here we report on the first on-chip digital microfluidic real-time PCR instrument for generating monodisperse microdroplet reactors, thermal cycling them for PCR, and detecting real-time amplification in the individual picoliter droplets.”
See Figure 3b, which shows the results of amplification with an average of 0.06 copies per droplet. As can be seen from the amplification curve at the right, amplification was monitored in real-time.
determining a total number of the individual subvolumes that contain an amplification product of the target nucleic acid;
See last paragraph on page 8474: “Examining Figure 3b and adjusting the droplet count to include three doublets and two each of the 3× and 4× droplets gives an equivalent total of 42 droplets, 2 of which showed obvious amplification…”.
Regarding claim 3, as discussed above, Beer disclosed PCR.
Regarding claim 4, as shown by the amplification curve in Figure 3b (which shows a plateau region), the amplification was performed until a “near-saturation concentration of the amplification product is produced in each subvolume containing the amplification product”.
Regarding claims 5 and 6, as shown by the amplification curve in Figure 3B, the amplification comprised multiple cycles (cycle number shown along the abscissa). See also page 8473, paragraph continuing from previous page, which outlines the thermal cycling protocol having 40 thermal cycles.
Regarding claims 7 and 8, see page 8473, last paragraph in first column: “The fluorescence microscope imaged droplets in a 300 × 500 µm section of the channel during the annealing phase of each cycle for real-time detection.”
Regarding claim 11, see last paragraph on page 8474: “Examining Figure 3b and adjusting the droplet count to include three doublets and two each of the 3× and 4× droplets gives an equivalent total of 42 droplets, 2 of which showed obvious amplification…”.
Regarding claims 12 and 13, see page 8473, first column, paragraph entitled “PCR Reagents”; Beer was amplifying “Vaccinia Western Reserve genomic DNA”.
Regarding claim 19, Beer made 10 pL droplets; see Table 1 for the experiment having an average of 0.06 copies of genomic DNA per singlet droplet.
Regarding claims 24 and 25, see page 8473, last paragraph in first column: “The fluorescence microscope imaged droplets in a 300 × 500 µm section of the channel during the annealing phase of each cycle for real-time detection.” See also the amplification curve in figure 3b.
Regarding claim 26, Beer determined Ct values; see Table 3, where the Ct values are shown.
Beer did not actually “determin[e] a starting number of copies of the target nucleic acid based on the determined total number”. This is because, as a proof-of-principle study, Beer already knew the concentration of DNA in the samples; see page 8473, first column, paragraph entitled “PCR Reagents”, where Beer states: “PCR quantification was performed to estimate DNA copy number of our stock template solution.”
However, Beer certainly had in mind to use the system he developed for quantitative purpose; see page 8475, first column, last full paragraph: “The excellent agreement between observed and Poisson predicted droplet amplification for the quantitated starting copy concentration across all dilutions shows the promise of picoliter droplets for quantitative PCR (see Table 2).”
Blow summarized the principle of digital PCR (page 869, second paragraph): “The quantitation in digital PCR is achieved by diluting the starting sample so that there is at most only one molecule in each well. After amplification, the number of wells containing the product is counted to determine the number of molecules in the starting sample.”
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to use the method of Beer for quantifying a target sequence in a sample (since Beer himself suggested this), and in doing so count the number of positive reactions to determine the number of molecules in the starting (i.e. diluted) sample, since Blow disclosed this was how the quantification by digital PCR worked.
Claim 9 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable Beer (Anal. Chem. 79:8471-8475 (2007), IDS ref) over Blow (Nature Methods 4(10)869-875 (2007)) as applied to claims 2-8, 11-13, 19 and 24-26 above, and further in view of Beck (J. Clin. Micro. 39(1):29-33 (2001)).
The disclosures of Beer and Blow have been discussed. While both Beer (see Table 1, noting four different “dilutions”) and Blow (page 869, second paragraph) mentioned the sample was diluted to achieve an appropriate concentration, neither mentioned that the number of target molecules in the diluted sample (as determined by the number of positive reactions) was multiplied by the dilution factor to determine the number of molecules in the original (undiluted) sample.
However, it was known in the art to correct for dilution. For example, Beck measured HIV proviral DNA copy number in patient samples using limiting-dilution PCR, where “[a] dilution resulting in one positive PCR out of five was considered to have one copy of HIV-1.” Beck states: “The number of HIV-1 copies/106 PBMC was calculated with the following equation: (66.7 × dilution factor × number of positive reactions)/(total number of reactions × volume of DNA template).”
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to modify the method suggested by the combined disclosures of Beer and Blow to take into account the dilution factor in order to quantify the number of target molecules in the original sample, as Beck demonstrates that such a correction was known and necessary when working with diluted samples.
Claims 14 and 15 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable Beer (Anal. Chem. 79:8471-8475 (2007), IDS ref) over Blow (Nature Methods 4(10)869-875 (2007)) as applied to claims 2-8, 11-13, 19 and 24-26 above, and further in view of Vogelstein (PNAS 96:9236-9241 (1999)).
The disclosures of Beer and Blow have been discussed. These references did not disclose applying the method to RNA.
Vogelstein, in one of the seminal publications on digital PCR, suggested (page 9240, left column): “Dig-PCR is as easily applied to RT-PCR products generated from RNA templates as it is to genomic DNA.” Vogelstein offered three scenarios where RNA could be analyzed (Table 1): chromosomal translocations, alternatively spliced products, and measuring changes in gene expression.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to apply the method suggested by the combined disclosures of Beer and Blow to RNA, since Vogelstein recommended applying digital PCR methods to RNA.
Claims 18, 20, 22 and 23 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable Beer (Anal. Chem. 79:8471-8475 (2007), IDS ref) over Blow (Nature Methods 4(10)869-875 (2007)) as applied to claims 2-8, 11-13, 19 and 24-26 above, and further in view of Hasson (WO 2008/005248 A2).
The teachings of Beer and Blow have been discussed. Neither reference taught a sample having at least 10 copies of nucleic acid, using at least 10% of sample, at least 50 droplets, at least 5000 droplets, or while flowing.
Hasson disclosed methods using Poisson statistics to quantify a nucleic acid in a sample based on the formation of aliquots. Hasson disclosed at paragraph [0093] (emphasis provided):
where simple detection of a rare nucleic acid is desired, enough low and/or single copy number aliquots are made of the sample to detect the nucleic acid in one of the aliquots. Where more quantitative information is needed, enough copies are made to provide reliable statistical information, e.g., to a given confidence value. In either case, this can include anywhere from 1 aliquot to 109 or more aliquots, e.g., 10, 100, 1,000, 10,000, 100,000, 1,000,000, 1,000,000,000 or more aliquots. There is no theoretical limit on the number of aliquots that can be made and assessed for a nucleic acid of interest according to the present invention, though there are practical considerations with respect to the throughput of the system and the size of the sample (the lower the throughput, the fewer aliquots can be analyzed in a given time; the larger the sample size the more aliquots can be made of the sample). Using microfluidic approaches, reagent usage (and concomitant reagent costs) can be minimized. By formatting the system to provide for continuous flow of sample and reagents, including, optionally, during amplification, the systems of the invention can greatly speed the process of searching many different samples for a nucleic acid of interest.
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 teachings of Beer and Blow by increasing the number of droplets formed (and thus the percentage of the sample used) into the claimed range in order to provide more reliable statistical information. It would additionally have been obvious to format the system for continuous flow, including during amplification, to “speed the process of searching many different samples for a nucleic acid of interest.”
Conclusion
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/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681