17778612
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 .
Priority
This application 17/778,612 filed on 05/20/2022 is a 371 national phase of PCT/US20/61467 filed on 11/20/2020, and claims the benefit of provisional U.S. Patent Application No. 62/938,138, filed on 11/20/2019.
The priority date of claim 8 and its dependent claims is determined to be 11/20/2019, the filing date of provisional U.S. Patent Application No. 62/938,138.
Status of Claims
Applicant’s amendments to claims filed 02/05/2026 in response to the Non-Final Rejection mailed 11/17/2025 are acknowledged.
Claim 63 is amended.
Claim 71 has been canceled.
New claim 117 is acknowledged.
Claims 8-19, 24, 61-64, 66-68, and 117 are pending and claims 8-18, 61-64, 66-68. and 117 are under examination.
Response to Remarks filed 02/05/2026
The amendments and arguments presented in the papers filed 02/05/2026 ("Remarks”) have been thoroughly considered. The issues raised in the Office action dated 11/17/2025 have been reconsidered as indicated.
New grounds of rejection necessitated by amendment are detailed below and this action is made FINAL.
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.
Claims 8-10, 13, 16-18, 61, 62, 66, 68, and 117 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kitayama et al. (Repeated fluorescence in situ hybridization by a microwave-enhanced protocol. 2006. Pathol Int. 56(9):490-3; on IDS filed 08/15/2023) in view of Shapiro et al. (Acetic acid treatment denatures DNA while preserving chromosomal morphology during the in situ hybridization procedure.1978. Experimental Cell Research.115(2): 411-414, on IDS dated 10/16/2024).
The rejection of claims 8-10, 13, 16-18, 61, 62, 66, and 68 are maintained.
Regarding claim 8, Kitayama teaches a method for cycles of fluorescence in situ hybridization (FISH), the method comprising probes that hybridize with genomic sequences in tissue sections and the use of an acid reagent (HCl) to strip the hybridized probes (p. 490, col 2 and p. 491, Table 1). Kitayama teaches washing the sample with HCl (i.e. contacting the cell with an acid reagent) (p. 491, col 1).
Kitayama does not teach the acid reagent is selected from acetic acid, formic acid, propionic acid, butyric acid, valeric acid, oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, and citric acid.
Shapiro teaches a method of in situ hybridization comprising denaturing of chromosomal DNA by acetic acid (p. 411, col. 2). Shapiro teaches testing multiple denaturants (p. 412, Table 1) in order to achieve efficiency and preservation of the target (p. 414, cols. 1 and 2).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kitayama and Shapiro to arrive at the instantly claimed invention. Shapiro teaches denaturing of chromosomal DNA (i.e. double stranded DNA) prior to hybridization however the denaturing of double stranded nucleic acid is equivalent to denaturing the probes hybridized to DNA (a double stranded nucleic acid) in Kitayama and achieves the same goal of separating the strands of nucleic acids. The modification would have entailed substituting the acetic acid denaturant of Shapiro as the acid reagent of Kitayama. One would have been motivated to make the substitution by optimizing for a denaturant that disrupted probe binding efficiently but with a milder treatment. Alternatively, the modification that would have entailed the addition of acetic acid in a further step. One would have been motivated by the weaker, milder nature of acetic acid in the treatment of double stranded nucleic acids. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 9, Kitayama teaches repeated stripping, i.e. repeated used of the acid reagent (p. 490, Abstract and p. 491, Table 1).
Regarding claim 10, Kitayama teaches removal of the first probe (p. 492, Fig. 1).
Regarding claim 13, Kitayama teaches contacting the cell with second probes that target second target nucleic acids different from the first probes (p. 492, Fig. 1).
Regarding claim 16, Kitayama teaches washing the sample with HCl (i.e. contacting the cell with an acid reagent) (p. 491, col 1). Kitayama teaches repeating the procedure indefinitely (p. 492, col. 2) with a step contacting the cell with the acid reagent in each cycle (p. 491, Table 1 and p. 492, Figs. 1-3), thus satisfying the requirement of disrupting hybridization between second probes and second target nucleic acids.
Regarding claim 17, Kitayama teaches repeated stripping using an acid reagent (p. 490, Abstract and p. 491, Table 1).
Regarding claim 18, Kitayama teaches repeating the procedure indefinitely (p. 492, col. 2)
Regarding claim 61, neither Kitayama nor Shapiro teach the acid reagent comprises 5-40% acid.
However, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Kitayama and Shapiro to arrive at the instantly claimed invention. The modification would have entailed using the acetic acid reagent of Shapiro at the claimed concentration. One would have been motivated to change the concentration in order to determine the optimum working concentration for the selected acid reagent in the modified method of Kitayama. Changes in concentration to discover optimum or workable ranges are recognized as routine experimentation and optimization within the level of ordinary skill in the art See MPEP 2144.05 IIA. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 62, Shapiro teaches the use of acetic acid for denaturation, i.e. separation of two strands of nucleic acid (p. 411, Summary).
Regarding claim 66, Kitayama teaches probes hybridized to genomic sequences, i.e. DNA (p. 490, col. 2)
Regarding claim 68, Kitayama teaches the sample is a tissue section (p. 491, Table 1), from a tumor (p. 490, col. 2).
The following new rejection has been added to address claim amendments adding claim 117 filed on 02/05/2026.
Regarding claim 117, Shapiro teaches the use of acetic acid as a reagent tor denaturing DNA (p. 411, Abstract).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kitayama and Shapiro to arrive at the instantly claimed invention. Shapiro teaches denaturing of chromosomal DNA (i.e. double stranded DNA) prior to hybridization however the denaturing of double stranded nucleic acid is equivalent to denaturing the probes hybridized to DNA (a double stranded nucleic acid) in Kitayama and achieves the same goal of separating the strands of nucleic acids. The modification would have entailed substituting the acetic acid denaturant of Shapiro as the acid reagent of Kitayama. One would have been motivated to make the substitution by optimizing for a denaturant that disrupted probe binding efficiently but with a milder treatment. Alternatively, the modification that would have entailed the addition of acetic acid in a further step. One would have been motivated by the weaker, milder nature of acetic acid in the treatment of double stranded nucleic acids. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Claims 11, 12, 14, 15, 63, 64, and 67 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kitayama et al. (Repeated fluorescence in situ hybridization by a microwave-enhanced protocol. 2006. Pathol Int. 56(9):490-3; on IDS filed 08/15/2023) in view of Shapiro et al. (Acetic acid treatment denatures DNA while preserving chromosomal morphology during the in situ hybridization procedure.1978. Experimental Cell Research.115(2): 411-414, on IDS dated 10/16/2024) as applied to claims 8-10, 13, 16-18, 61, 62 and 66-68 above, and further in view of Codeluppi et al. (Spatial organization of the somatosensory cortex revealed by osmFISH. 2018. Nat Methods. 15: 932–935 and Methods p.1-2, on IDS dated 08/15/2023).
The rejection of claims 11, 12, 14, 15, 63, 64, and 67 are maintained.
Regarding claims 11 and 12, neither Kitayama nor Shapiro teach two or more first probes hybridized to two or more first target nucleic acids (claim 11); or wherein each of the first target nucleic acids is labeled by hybridization to the first probes, and
wherein the label on each first target nucleic acid is distinguishable from the label on the other first target nucleic acid(s) hybridized to the first probes (claim 12).
Codeluppi teaches three different probe sets targeting three transcripts added at one time (Methods, p. 1, col. 2), as encompassed by the two or more first probes hybridized to two or more first target nucleic acids of claim 11.
Codeluppi further teaches multiple transcripts targeted at each round of hybridization, distinguished by fluorescent color, with visualization upon binding
of fluorescently labeled DNA probes (p. 932, col. 1). Codeluppi states that their method has the ability to map a large number of markers simultaneously (p. 932, col. 1)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kitayama and Shapiro with Codeluppi to arrive at the instantly claimed invention. The modification would have entailed using the probe set of Codeluppi in the method of Katayama during the first hybridization cycle. One would have been motivated by the ability provided by the probe sets of Codeluppi to acquire more information about target nucleic acids in single sections that might be limited, a concern of Kitayama (p. 490, col.2). There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claims 14 and 15, neither Kitayama nor Shapiro teach two or more second probes hybridized to two or more second target nucleic acids (claim 14); or wherein each of the second target nucleic acids is labeled by hybridization to the second probes, and wherein the label on each second target nucleic acid is distinguishable from the label on the other second target nucleic acid(s) hybridized to the second probes (claim 15).
Codeluppi teaches a method of fluorescence in situ hybridization (FISH), performing multiple rounds of hybridizations (p. 932, col. 1). Codeluppi teaches three different probe sets targeting three transcripts added at one time (Methods, p. 1, col. 2), as encompassed by the two or more second probes hybridized to two or more second target nucleic acids of claim 14.
Codeluppi further teaches multiple transcripts targeted at each round of hybridization, distinguished by fluorescent color, with visualization upon binding
of fluorescently labeled DNA probes (p. 932, col. 1).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kitayama and Shapiro with Codeluppi to arrive at the instantly claimed invention. The modification would have entailed using the probe set of Codeluppi in the method of Katayama during the second hybridization cycle. One would have been motivated by the ability provided by the probe sets of Codeluppi to acquire more information about target nucleic acids in single sections that might be limited, a concern of Kitayama (p. 490, col.2). There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claims 63 and 64, Kitayama teaches an acid reagent for probe stripping (p. 490, col 2 and p. 491, Table 1). Shapiro also teaches an acid reagent for denaturing double stranded nucleic acids (p. 413, Table 1).
Neither Kitayama nor Shapiro teach the acid reagent further comprises a salt (claim 63); or the acid reagent further comprises 1X to 13X saline sodium citrate (SSC).
Codeluppi teaches testing different stringency conditions (temperature, formamide and salts concentration) and stripping reagents (DNase, formamide and salt concentration) (Methods, p. 1, col. 1) and optimizing their FISH method with extensive testing of stripping reagents (p. 932, cols 1 and 2).
Codeluppi teaches stripping a probe with 65% formamide in 2× SSC (Methods p. 1, col. 2). Thus, Codeluppi teaches elements that satisfy the requirements of a salt (claim 63) and SSC between 1x and 13x concentration (claim 64).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kitayama and Shapiro with Codeluppi to arrive at the instantly claimed invention. The modification would have entailed using the SSC salt of Codeluppi with the acid reagent of Kitayama. Codeluppi recognized the importance of testing and optimizing reagents and concentrations for stripping steps of FISH. Shapiro similarly recognized multiple reagents could perform the same task of denaturing double stranded nucleic acids; including an reagent comprising an acid reagent or SSC salt (p. 413, Table 1). One would have been motivated to add the SSC salt of Codeluppi in order to optimize the stripping ability of the acid reagent. Changes in concentration to discover optimum or workable ranges are recognized as routine experimentation and optimization within the level of ordinary skill in the art See MPEP 2144.05 IIA. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 67, Kitayama teaches DNA-RNA hybridization and the use of DNA probes (p. 490, col. 1). However, Kitayama teaches targets are genomic (DNA) (p. 490, col. 2) and neither Kitayama nor Shapiro teach the target nucleic acids are RNA.
Codeluppi teaches a method of FISH with RNA as targets (p. 932, col. 1)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Kitayama and Shapiro with Codeluppi to arrive at the instantly claimed invention. The modification would have entailed RNA as the target nucleic acids. Kitayama recognized RNA as target nucleic acids and the use of DNA probes as used in Kitayama for hybridizing to RNA targets and the use of targets in FISH was well known in the art (Kitayama p. 490, col. 1). One would have been motivated in order to perform the method on RNA in the cell. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Response to Arguments against Claim Rejection - 35 U.S. C § 103
Claims 8-10, 13, 16-18, 61, 62,66, and 68
The response asserts that the method of Kitayama conducts several pre-treatment sample preparation steps before the probing/ staining/stripping/rehybridization sequence, citing Table 1 of Kitayama, and provides Exhibit A, a FISH protocol manual from Agilent Technologies published in 2015 in support of the assertion that these pre-treatment steps are well-known to those skilled in the art of in situ hybridization (p. 9-10). The response further asserts that Shapiro's use of acetic acid to denature chromosomal DNA is not equivalent to denaturing the probes hybridized to DNA (a double stranded nucleic acid) in Kitayama. Applicant argues that in Kitayama, DNA denaturation and probe stripping are different steps that use different reagents and different conditions. And further, that if they were equivalent, Kitayama could have simply repeated its denaturation step (p. 10-11).
The response asserts that if one skilled in the art were to have considered combining Kitayama and Shapiro, at best, one skilled in the art would have considered replacing Kitayama's denaturation step using formamide/SSC with Shapiro's acetic acid denaturation step. The response asserts that there is no evidence of record to show that one skilled in the art would have been motivated to substitute Shapiro's acetic acid for the HCl in Kitayama's probe-stripping step (p. 11).
Applicant's arguments have been fully considered but are not persuasive.
It is acknowledged that the pre-treatment steps of Kitayama are well-known in the art of in situ hybridization. However, these steps are encompassed by the open claim language “comprising” found in the instant claims.
Regarding the assertion that, if the steps of DNA denaturation and probe stripping of Kitayama were equivalent Kitayama could have simply repeated its denaturation step, Kitayama uses an acid reagent for probe stripping in repeated cycles of in situ hybridization and one of skill in the art would have been motivated to find an alternative acid reagent that functions by the same mechanism (i.e., breaking down hydrogen bonds between base pairs in nucleic acids), but with different properties that further improve the multiple cycles of hybridization. The acetic acid of Shapiro would have been known by one in the art to be a weaker acid than the hydrochloric acid of Kitayama and would have been predicted to be less harsh on the target nucleic acids.
Although the DNA denaturation and probe stripping of Kitayama are different steps with different reagents and conditions, they are equivalent in that they perform the same function of separating nucleic acids. However, this equivalence would not limit one of skill in the art to considering only choosing between reagents and conditions taught by a single method (here that of Kitayama).
Further, it is noted that the scope of claim 8 does not exclude the use of low concentration hydrochloric acid in an additional step.
The response further submits that one skilled in the art would not have reasonably expected success in using acetic acid in Kitayama' s process, because Shapiro teaches that acetic acid has significant limitations as a denaturant in its own methods. And therefore, even if the effectiveness of acetic acid were a proxy for effectiveness at probe
stripping, Applicant submits that one skilled in the art would not have reasonably
expected success in using acetic acid due to its poorer performance compared to other methods tested in Shapiro (p. 11).
Applicant's arguments have been fully considered but are not persuasive.
One of skill in the art would reasonably have expected success in using acetic acid because the method of Kitayama teaches the use of an acid reagent for probe stripping. The acetic acid reagent of Shapiro functions by the same mechanism as the acid reagent of Kitayama, namely breaking down hydrogen bonds between base pairs in nucleic acids. In addition, as noted by the applicant, Shapiro teaches that acetic acid performed better than HCl (Remarks p. 11).
The response asserts that the evidence of record does not show a reason that one skilled in the art would have been motivated to substitute the HCl used for probe stripping in Kitayama with the acetic acid used for denaturing DNA in Shapiro, or that one would have reasonably expected success in doing so (p. 11).
Applicant's arguments have been fully considered but are not persuasive.
One of skill in the art would have known that both hydrochloric acid and acetic acid function by breaking down hydrogen bonds between base pairs. Both the hybridized probes of Kitayama and the chromosomal DNA of Shapiro would have been recognized as double stranded nucleic acids that would be separated by treatment with acid. One of skill in the art would also have known that acetic acid is a weaker acid than hydrochloric acid and would have been motivated to substitute acetic acid for the hydrochloric acid in the probe stripping step for the predicted benefit of a less disruptive removal of the hybridized probes. The method of Kitayama teaches repeating the hybridization cycle and a less disruptive probe stripping would be expected to improve the efficacy of the repeated cycles.
Further, as noted above, the scope of the claims does not exclude the addition of a step using acetic acid rather than a substitution of acetic acid. One of skill in the art would have been motivated to add an acetic acid treatment for the milder separation of the probes in a possible step-wise treatment.
Claims 11, 12, 14, 15, 63, 64, and 67
The response asserts that Codeluppi does not remedy the deficiencies of Kitayama and Shapiro in the rejection of claims 8-10, 13, 16-18, 61, 62, 66, and 68 (p. 12).
Applicant's arguments have been fully considered but are not persuasive.
No specific argument has been presented against the rejections. Responses to the arguments against Kitayama and Shapiro are presented above.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
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/JESSICA GRAY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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