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 .
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.
Claims 1, 2, 5-7, 11-13, 30-32, 35-38, 40-43 are rejected under 35 U.S.C. 103 as being unpatentable over Hutchison et al. (US 2012/0302448, IDS reference) in view of Kartalov et al. (US 2015/0057178, IDS reference).
With regard to claim 1 Hutchison disclosed:
A method for performing a digital assay,
See, e.g., paragraph [0016]: “Methods of the invention involve forming sample droplets containing target nucleic acid. Ideally, methods of the invention comprise forming droplets for digital PCR.”
comprising: (a) partitioning into a plurality of partitions (i) three or more nucleic acid targets and (ii) a set of nucleic acid probes comprising a first nucleic acid probe, a second nucleic acid probe, and a third nucleic acid probe;
See, e.g., paragraph [0006]: “Digital PCR (dPCR) is an alternative quantitation method in which dilute samples are divided into many separate reactions.”
See, e.g., paragraph [0009]: “Microfluidic droplets for multiplex analysis according to the invention contain a plurality of probes that hybridize to amplicons produced in the droplets. Preferably, the droplet contains two or more probes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 500, or more probes…The plurality of probes can include one or more groups of probes at varying concentrations. The one or more groups of probes can include the same detectable label which will vary in intensity upon detection, due to the varying probe concentrations. ”
See, e.g., paragraph [0150]: “One method of the invention involves performing higher-plex assays with a single probe color (i.e. fluorophore). As described above, probe fluorescent intensities can be adjusted by a variety of means such that each intensity level uniquely identifies a DNA target. For example, targets T1, T2, T3, and T4 might be uniquely identified by intensity levels I1, I2, I3, and I4.”
See, e.g., figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method. Probes for TERT, RNaseP and E1a were all labeled with the same fluorophore (VIC).
(b) amplifying the three or more nucleic acid targets in the partitions;
See, e.g., figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method.
(c) generating N signals from the set of nucleic acid probes, each of which N signals corresponds to the presence of a unique combination of nucleic acid targets of the three or more nucleic acid targets in a partition of the plurality of partitions;
See, e.g., figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method. As shown in figure 5C, the three probes labeled with VIC (TERT, RNaseP and E1a) resulted in 3 different signals, each corresponding to a different probe, and each characterized by a different signal intensity.
and (d) detecting the N signals in a single optical channel;
See, e.g., figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method. As shown in figure 5C, the three probes labeled with VIC (TERT, RNaseP and E1a) were all detected in a single optical channel (the VIC channel).
With regard to claim 2, see figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method. As shown in figure 5C, the three probes labeled with VIC (TERT, RNaseP and E1a) resulted in 3 different signals.
With regard to claims 5 and 7 see paragraph [0150]: “One method of the invention involves performing higher-plex assays with a single probe color (i.e. fluorophore). As described above, probe fluorescent intensities can be adjusted by a variety of means such that each intensity level uniquely identifies a DNA target. For example, targets T1, T2, T3, and T4 might be uniquely identified by intensity levels I1, I2, I3, and I4.” See also figure 5 and paragraphs [0120]-[0125], which describes an example in which 5 targets were amplified (TERT, RNaseP, E1a, SMN1 and SMN2) in a droplet digital PCR method. As shown in figure 5C, the three probes labeled with VIC (TERT, RNaseP and E1a) resulted in 3 different signals, each corresponding to a different probe, and each characterized by a different signal intensity.
With regard to claims 11-13, see paragraph [0017]: “The targets are then amplified in the droplets. Any method known in the art may be used to amplify the target nucleic acids either linearly or exponentially.” See also multiple references to droplet digital PCR in the discussion above. PCR comprises nucleic acid extension (as do linear amplification methods).
While Hutchison disclosed different probes having the same fluorescent label present in the mixture at different concentrations, resulting in different signal intensities (e.g. paragraph [0150), Hutchison did not specifically disclose the concentrations in an X, 2X, 4X, 8X as recited in claims 1 and 6.
However, Hutchison did disclose (paragraph [0150]): “…the maximum number of intensity levels possible for unique identification of targets is related to the resolution of the different intensity levels…”. Thus, probe concentrations would need to differ sufficiently to be resolved as discrete signals (as seen, e.g., in figure 5C).
Kartalov also disclosed methods of multiplexing based on (among other things) signal intensity of probes having the same fluorophore (see Example 3, paragraphs [0184]-[0185]). Kartalov disclosed: “As shown in Table 17, each analyte was encoded as a single value of fluorescence intensity in a single color. In this case, as described elsewhere in this disclosure, the coding scheme is non-degenerate by design. This is achieved by assigning an intensity value to each analyte that is equal to the cumulative intensity values for the prior analytes, plus one…The fluorescence signals generated by the respective probes in positive-control end-point PCR reactions were measured and used to calculate the probe concentrations that would produce lx, 2x, and 4x signal intensities.”
In addition, Kartalov stated (paragraph [0156]): “The methods described in this disclosure are compatible with a variety of amplification methods, including quantitative PCR (qPCR) methods, end point PCR methods, reverse transcriptase PCR, and digital PCR methods. Digital PCR methods (e.g., DROPLET DIGITAL (BIORAD) and DYNAMIC ARRAY (FLUIDIGM)) produce highly sensitive quantification of polynucleotide copy numbers. The methods provided herein can be easily integrated into these systems to significantly expand their throughput by allowing multiplexing in a droplet or a dynamic array.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the instant application to vary the concentrations of the different probes when practicing the method of Hutchison to provide for appropriate resolution between probe intensities. Since Kartalov had already disclosed the concept of having signal intensities of a single color vary among targets in a fashion such that the intensity of the next probe equaled the cumulative intensities of the previous probes plus 1 (e.g. 1x, 2x, 4x, 8x), it would have been obvious to use this encoding scheme when setting the probe concentrations in the practice of Hutchison’s method (thus arriving at probe concentrations in a 1x, 2x, 4x, 8x progression). Moreover, Kartalov expressly suggested applying his methods to a digital PCR regime.
As claims 30-31 are simply broader versions of claims 1 and 2, and claim 32 is simply the result of the natural progression to a fourth probe (see Hutchison paragraph [0150] discussing four targets with four intensity levels), these claims are obvious over the combination of Hutchison and Kartalov for the same reasons discussed above.
The limitations of claims 36-38 and 40-43 follow from the discussion of claims 1, 2, 5-7 and 11-13 above, and are therefore obvious for the same reasons.
Claims 14-18, 23-26 and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Hutchison et al. (US 2012/0302448, IDS reference) in view of Kartalov et al. (US 2015/0057178, IDS reference) as applied to claims 1, 2, 5-7, 11-13, 30-32, 35-38, 40-43 above, and further in view of Jouvenot (US 2014/0171341, IDS reference).
The disclosures of Hutchison and Kartalov have been discussed. Both discussed multiplexing based on different probe intensities for different targets, where the different probe intensities were achieved by varying the concentration of each probe. Neither of these documents disclosed using probes with different fluorophores, but detectable in a single optical channel (as recited in claim 26). Neither of these documents disclosed achieving different probe intensities by using probes wherein each probe per se had a different fluorescence intensity, achieved either by (i) using a different number of fluorophores (each having the same intensity) on each probe (as recited in claims 15 and 39), or (ii) using different fluorophores (each having a different intensity) on each probe (as implied in claim 16).
Jouvenot also disclosed multiplexing based on probe intensity. Specifically, Jouvenot disclosed (paragraph [0084], emphasis provided):
Fluorophores 92, 94, which may be the same or different, create detectable but distinguishable signals in the same channel, allowing multiplexing in that channel. The signals may be distinguishable because an aspect of the fluorescence is different for one fluorophore than for the other fluorophore(s). For example, the intensity associated with one fluorophore, following reaction, may be lower or higher than the intensity(ies) associated with the other fluorophore(s). In some embodiments, one probe may be labeled with a different number of fluorophores than the other probe, and/or the probes may be located in slightly different local environments, creating a different level of fluorescence for each probe following reaction. Alternatively, or in addition, both probes may be labeled with the same number of fluorophores (e.g., one fluorophore), but there may be more or less of one probe than the other in the sample, so that a greater or smaller signal is created when the reactions have occurred. In some cases, the fluorophores themselves might be different, with one more or less intrinsically fluorescent than the other (e.g., due to differences in extinction coefficient, quantum yield, etc.), so long as each fluorophore can be detected in the same channel.
The language “…one probe may be labeled with a different number of fluorophores than the other probe…” implies that, in the embodiment of using a different number of fluorophores to achieve different signal intensities, each of said fluorophores itself has the same intensity. If the fluorophores already had different intensities, there would be no reason to use different numbers of such fluorophores.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the instant application to modify the method suggested by the combined disclosures of Hutchison and Kartalov by, instead of using a different probe concentrations to achieve different intensities (a technique also mentioned by Jouvenot; see above), using either different numbers of fluorophores having the same intensity on each probe (thus meeting the limitations of claims 15 and 39), or using different fluorophores having different intrinsic intensities on each probe (thus meeting the limitations of claims 16). Either technique would have been obvious because Jouvenot taught them as alternatives to varying probe concentration in order to achieve different signal intensities for different probes, for the purpose of multiplexing in a single optical channel. The rationale to use signal intensities of Y, 2Y, 4Y and 8Y is the same as the rationale to use probe concentrations of X, 2X, 4X and 8X as discussed in the rejection of claim 1 (i.e. based on the teachings of Kartalov).
Conclusion
No claims are free of the prior art.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMUEL C WOOLWINE whose telephone number is (571)272-1144. The examiner can normally be reached 9am-5:30pm.
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/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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