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 .
Response to Amendment
The amendment filed 03/24/2026 is acknowledged. Regarding the Office action mailed 01/05/2026:
The objections to the specification are withdrawn in view of the substitute specification filed 03/24/2026.
The non-statutory double patenting rejection(s) is/are withdrawn in view of the terminal disclaimer filed 03/24/2026.
The rejections under 35 USC 103 are maintained and reiterated below. Applicant’s arguments are addressed following the rejections.
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) 37, 39 and 40 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dobosy (BMC Biotechnology 11:80 (2011)) in view of Knoll (US 2008/0085509) and Egrie (US 7,304,150).
Dobosy disclosed a method for conducting PCR using blocked primers. The blocking moiety was joined at the 3’ end of the primers via a ribonucleotide. Upon hybridization to the target sequence to be amplified, the ribonucleotide was cleaved by RNase H2, releasing the blocking moiety and rendering the primers capable of amplification. See Fig. 1. Regarding claim 40, Dobosy used the method on cDNA; see page 5, left column, section titled “Studies of the specificity of rhPCR using mammalian cDNA”. cDNA is by definition produced by a reverse transcriptase mediated template dependent primer extension.
Dobosy did not disclose the particular order in which the components of the PCR reaction were combined.
Knoll disclosed (paragraph [0060]): “In preparing the PCR reaction mixture, the various constituent components may be combined in any convenient order.”
Egrie disclosed (column 15, lines 43-52): “The typical PCR reaction mix contained: 4 µl each of forward and reverse primers (5 pmol/µl), 1 µl template (25 ng), 10 µl of 5×LP buffer (100 mM Tricine pH 8.7/25% glycerol/425 mM KOAc), 10 µl dNTP stock (1 mM each of dATP, dTTP, dCTP, dGTP), 0.8 µl rtTh polymerase (Perkin Elmer; 2.5 U/µl), and 2 µl Vent polymerase (NEB; 0.01 U/µl after 1:100 fresh dilution in 1×LP buffer). H2O was added to bring the final volume to 50 µl. All the components were added together in the order shown…”. Note that the first two components combined were the primers and the template.
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 combine the template and primers prior to adding the RNase H2 in practicing Dobosy’s method, since this would merely represent one of a finite number of possible orders of combining Dobosy’s reaction components, Knoll disclosed that PCR components could be combined in any convenient order, and Egrie demonstrates that it was known to combine the primers and templates first.
Claim(s) 37, 39 and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Shi (Human Mutation 28(2):131-136 (2007)) in view of Knoll (US 2008/0085509) and Egrie (US 7,304,150).
Shi disclosed a method for conducting PCR using blocked primers to detect mutations. The blocking moiety was a dideoxynucleotide at the 3’ end of the primers. Upon hybridization to the corresponding target sequence to be amplified, dideoxynucleotide was removed from the primers by the polymerase by pyrophosphorolysis. Shi disclosed using a pair of such blocked primers, one corresponding to the wild-type sequence, one corresponding to the mutation. See Fig. 1. Regarding claim 42, note that Shi disclosed the use of the primers with the mismatched template (Fig. 1, middle and right scenarios), in which case the blocked primers comprised a non-template nucleotide sequence.
Shi did not disclose the particular order in which the components of the PCR reaction were combined.
Knoll disclosed (paragraph [0060]): “In preparing the PCR reaction mixture, the various constituent components may be combined in any convenient order.”
Egrie disclosed (column 15, lines 43-52): “The typical PCR reaction mix contained: 4 µl each of forward and reverse primers (5 pmol/µl), 1 µl template (25 ng), 10 µl of 5×LP buffer (100 mM Tricine pH 8.7/25% glycerol/425 mM KOAc), 10 µl dNTP stock (1 mM each of dATP, dTTP, dCTP, dGTP), 0.8 µl rtTh polymerase (Perkin Elmer; 2.5 U/µl), and 2 µl Vent polymerase (NEB; 0.01 U/µl after 1:100 fresh dilution in 1×LP buffer). H2O was added to bring the final volume to 50 µl. All the components were added together in the order shown…”. Note that the first two components combined were the primers and the template.
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 combine the template and primers prior to adding the polymerase in practicing Shi’s method, since this would merely represent one of a finite number of possible orders of combining Shi’s reaction components, Knoll disclosed that PCR components could be combined in any convenient order, and Egrie demonstrates that it was known to combine the primers and templates first.
Claim(s) 41 and 43 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dobosy (BMC Biotechnology 11:80 (2011)) in view of Knoll (US 2008/0085509) and Egrie (US 7,304,150) as applied to claims 37, 39 and 40 above, and further in view of Maryanski (Molecular Immunology 36:745-753 (1999)).
The teachings of Dobosy, Knoll and Egrie have been discussed. None of these references taught or suggested applying Dobosy’s method to template derived from a single lysed cell, or using the method to prepare a T-cell receptor repertoire library.
Maryanski prepared a T-cell receptor repertoire library by obtaining single cells from antigen-stimulated mice by PCR; see abstract: “Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice.” See also page 746, section 2.2. While Maryanski did not specifically state that the T-cells were “lysed”, it is implicit that this would have occurred, at the very least during the PCR itself, since the genomic DNA of the cell would not have otherwise been accessible to the primers, polymerase and other components of the PCR reaction.
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 Maryanski by using the blocked primers of Dobosy (along with the obvious order of combining the reaction components suggested by the teachings of Knoll and Egrie), because Dobosy taught (abstract): “rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification.”
Claim(s) 48 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dobosy (BMC Biotechnology 11:80 (2011)) in view of Knoll (US 2008/0085509), Egrie (US 7,304,150) and Maryanski (Molecular Immunology 36:745-753 (1999)) as applied to claims 41 and 43 above, and further in view of Cram (Methods in Cell Science 24:1-9 (2002)).
The teachings of Dobosy, Knoll, Egrie and Maryanski have been discussed. In short, it would have been obvious to use Dobosy’s blocked primers to conduct PCR on single cells when practicing the method of Maryanski, and to combine the single cells/lysates with the components of the PCR reaction in any convenient order as disclosed by Knoll, including combining the template (i.e. sample) with the primers first (as disclosed by Egrie). The collective teachings of the references to not explicitly indicate conducting the steps of combining sample, primers, and RNase H2 in “droplets” as recited in claim 48.
However, Maryanski’s method did employ flow cytometry to sort single cells.
Cram discussed the basic process of sorting cells with a flow cytometer (section 2.8, beginning on page 7, and see figure 15). In short, cells are sorted by deflecting droplets from the flow cytometer into collection vessels.
Therefore, in sorting cells in the method of Maryanski, then adding the primers of Dobosy, followed by the addition of RNase H2, to the droplets collected, one would have arrived at the method of claim 48.
Response to Arguments
Applicant's arguments filed 03/24/2026 have been fully considered but they are not persuasive. Applicant argues (emphasis provided):
“…Claim 37 recites the steps of (a) combining a blocked primer with a template nucleic acid component to produce a blocked primer reaction mixture and (b) adding an enzyme to the blocked primer reaction mixture to unblock the blocked primer of the blocked primer reaction mixture to produce an activated primer reaction mixture.
These are two separate and distinct steps that must be performed in this order, as the blocked primer reaction mixture must first be produced before the enzyme can be added to it. By forming the blocked primer reaction mixture first without adding an enzyme, additional reactions may be performed while the primers remain blocked. The significance of this purpose is demonstrated in the instant specification (see, e.g., Example 2).”
This argument is not persuasive. Firstly, the claims do not require performing “additional reactions” while the primers remain blocked, and therefore the “significance” of the recited order is not captured by the claims. Indeed, the claims merely require that primers are added to a template nucleic acid (which, in the methods of Dobosy or Shi, would be the “sample”) before adding enzyme (which, in the methods of Dobosy or Shi, would be the RNase H2 or polymerase, respectively). There need be no intermediate steps between these events, such as performing “additional reactions”, or even any delay in adding the subsequent reaction components. Secondly, the prior art teaches that components may be added in any convenient order, including, in the case of Dobosy, adding the primers to the sample before or after adding the RNase H2. Or, in the case of Shi, adding the primers to the sample before or after adding the polymerase, which is the enzyme that removes the dideoxynucleotide from the primers. Because the prior art (Knoll) taught that components of a PCR could be added “in any convenient order”, adding the primers to the sample before adding the respective enzymes in Dobosy and Shi was obvious and within the purview of the public. By granting the claims as currently written, options for the order of combining reactants when practicing the methods of Dobosy or Shi, options obvious and available to the public, would be taken away from the public. As discussed in MPEP 2145(II), "The fact that appellant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Here, Applicant points to an advantage in the claimed order of combining the reactants, that being the ability to perform “additional reactions” while the primers remain blocked. However, granting a patent on the discovery of an unknown but inherent function "would remove from the public that which is in the public domain by virtue of its inclusion in, or obviousness from, the prior art." In re Wiseman, 596 F.2d 1019, 201 USPQ 658 (CCPA 1979).
Applicant argues:
“…Dobosy fails to teach the steps of (a) combining a blocked primer with a template nucleic acid component to produce a blocked primer reaction mixture and (b) adding an enzyme to the blocked primer reaction mixture to unblock the blocked primer of the blocked primer reaction mixture to produce an activated primer reaction mixture.
…in the method of Dobosy, there is no reason to separate the addition of the enzyme from the addition of the other reagents since it is desirable in Dobosy's method to unblock and activate a primer as soon as it binds to the target DNA.”
As to “separating” the addition of the enzyme from the addition of the other reagents, the claims simply require a particular order of combining the components of the reaction, and while the method of Dobosy does not “need” to combine the sample with the primers before combining the sample with the RNaseH2, Knoll taught combining the reactants of a PCR reaction “in any convenient order”.
Applicant argues:
“…Knoll provides no guidance regarding reaction mixtures comprising blocked primers. In fact, Knoll is silent on any blocked primers, much less blocked primers comprising an enzymatically labile blocking moiety. Accordingly, as Knoll is directed to unrelated PCR methods, Knoll provides no reason why a skilled person would separately conduct the instantly claimed steps involving blocked primers and an enzyme used to remove the blocking moiety of the blocked primers.”
Applicant makes a similar argument regarding the disclosure of Egrie. These arguments are not persuasive. One of ordinary skill in the art would have understood that what Knoll teaches regarding combining reactants in any convenient order, and what Egrie teaches about combining sample with primers before adding enzyme, would apply to any PCR, including those using blocked primers as in the methods of Dobosy and Shi.
Applicant argues:
“the Office asserts that Shi discloses a method for conducting PCR using blocked primers to detect mutations wherein the blocking moiety is a dideoxynucleotide and the dideoxynucleotide is removed by pyrophosphorolysis. The Office asserts that Shi does not disclose the particular order in which the components of the PCR reaction are combined, for which the Office turns to Knoll and Egrie. The Office contends that it would have been obvious to combine the template and primers prior to adding the RNase H21 in practicing Shi's method since Knoll discloses that PCR components can be combined in any convenient order and Egrie demonstrates that it was known to combine the primers and templates first.”
Applicant continues:
“Pyrophosphate is not an enzyme. Shi is silent on any enzymatically labile blocking moiety. Accordingly, there is no teaching or suggestion in Shi of adding an enzyme to the blocked primer reaction mixture to enzymatically remove the enzymatically labile blocking moiety of the blocked primer.”
This argument is not persuasive and is inaccurate. See footnote 1 above. The polymerase (an enzyme) is what removes the dideoxynucleotides. While the process requires the presence of pyrophosphate as a reactant, the polymerase removes the dideoxynucleotides enzymatically.
Applicant argues:
“Further, as discussed above, Knoll and Egrie are silent on any blocked primers, much less
blocked primers comprising an enzymatically labile blocking moiety and thus fail to make up for the deficiencies in Shi.”
This argument has already been addressed above in the context of the Dobosy rejection, and is not persuasive for the same reaons.
The remainder of Applicant’s arguments rely on the same principles as discussed above, and are similarly not persuasive.
Conclusion
THIS ACTION IS MADE FINAL. 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 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
1 To clarify, the rejection indicated “polymerase”, not “RNase H2”. In Shi’s method, the polymerase enzymatically removes the dideoxynucleotides from the ends of the primers in the presence of pyrophosphate. The term “pyrophosphorlysis” does not mean the pyrophosphate removes the dideoxynucleotide.
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