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 is a continuation of 17/335,931, filed June 1, 2021, which is a continuation of PCT/EP202-065090, filed May 29, 2020. This application claims priority to foreign applications: UNITED KINGDOM 1907764.3, 1907752.8, 1907779.1, and 1907772.6, all filed on May 31, 2019.
Claim Status/Action Summary
Claims 51-54 and 57-72 are under examination. Claim 72 is newly added in the response filed March 23, 2026. No other claims are currently pending in this application.
This action is in response to the papers filed March 23, 2026.
Any objections and rejections not reiterated below are hereby withdrawn.
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.
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.
Claims 51-52, 54, 57-58, 60-71, and newly added claim 72 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., “Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing” Anal. Chem. 2018, 90, 12180-12186, in view of Dunaway et al., US 2018/0142286 A1, published May 24, 2018.
This rejection has been updated as necessitated by the amendment adding new claim 72.
Regarding claim 51, Zhang teaches a nucleic acid detection system comprising padlock probes comprising target binding sites, identifying barcode sequences, and a universal pool of reporter probes comprising two different fluorescent reporter probes (Zhang et al., Figure 1).
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Zhang teaches direct detection of the rolling circle amplification product (and decoding of the target-specific barcode) with detectably labeled universal reporter probes by quantifying the ratio of fluorescent signals corresponding to the two universal reporter probes. Zhang teaches that this system for multiplex detection of nucleic acid barcode sequences may be expanded to a greater number of distinguishable fluorescence ratios (Zhang et al., page 12184, column 1, paragraph 2) by adding more barcode copies, shortening the length of the barcodes, and/or adding more distinct barcodes with their own fluorophore-labeled probes (Zhang et al., page 12185, column 2, paragraph 3).
Zhang does not teach an expanded encoding system for recognizing a greater number of potential barcodes comprises: a) a first detection probe comprising a first domain sequence of the padlock barcode (i.e. hybridizing to the rolling circle product) and an overhang portion that is complementary to a universal detection probe and b) a second detection probe comprising a second domain sequence of the padlock barcode (i.e. hybridizing to the rolling circle product) and an overhang portion that is complementary to a universal detection probe, wherein the first and second domain sequences are capable of initiating a strand displacement reaction (i.e. are partially overlapping).
However, Dunaway et al. teach probe sets comprising primary “sequencing probes” that are analogous to the “detection probes” of the present disclosure. The “sequencing probes” taught by Dunaway et al. comprise a “target binding domain” that hybridizes to a target sequence (i.e. is analogous to a first or second domain sequence) and a “barcode domain” that hybridizes to fluorescently-labeled reporter probes (i.e. is analogous to a first or second overhang sequence) (Dunaway et al., figure 3).
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Dunaway et al. further teach that a series of sequencing probes (i.e. detection probes) can be used to sequentially characterize a target sequence in a “sequencing cycle” (Dunaway et al., figure 14). Finally, Dunaway et al. teach that the “sequencing cycle” of figure 14 can be repeated any number of times and that the sequencing probes can bind to the target nucleic acid at a position that overlaps the position at which the first sequencing probe was bound during the first sequencing cycle (i.e. the sequencing probes overlap and are capable of initiating a strand displacement reaction) (Dunaway et al., paragraph 0272).
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the padlock probe system for ratiometric fluorescence detection/decoding of a small number of multiplexed padlock probes using fluorescently-labeled reporter probes that directly bind to the rolling circle amplification product, taught by Zhang et al., with the teachings of Dunaway et al. comprising “sequencing probes” (i.e. detection probes) that hybridize to a target sequence that can overlap between two or more sequencing probes and fluorescently labeled, universal “reporter probes” that hybridize to an overhang domain of the “sequencing probes”. The ordinary artisan would have been motivated to use the detection probes and reporter probes taught by Dunaway et al. to detect the multiplexed padlock barcodes taught by Zhang et al. rather than the direct binding of reporter probes to the barcodes, as taught by Zhang et al., because of the suggestion of Zhang et al. that the system for multiplex detection of nucleic acid barcode sequences comprising ratiometric detection of fluorescence signals may be expanded to a greater number of distinguishable fluorescence ratios (Zhang et al., page 12184, column 1, paragraph 2) by adding more barcode copies, shortening the length of the barcodes, and/or adding more distinct barcodes with their own fluorophore-labeled probes (Zhang et al., page 12185, column 2, paragraph 3). Further, Dunaway teaches that given a first or second domain sequence as short as 6 nucleotides and detection probes labeled with as few as two fluorophores allows for discrimination between 512 distinct target sequences (Dunaway et al., figure 3). Both Zhang et al. and Dunaway et al. teach nucleic acid systems for detecting specific target systems using combinations of fluorescent signals localized to the target nucleic acid. Therefore, the ordinary artisan would have had a reasonable expectation that the detection probe and reporter probe system for detecting hundreds of distinct target sequences using combinations of fluorescent signals encoded in the overhang domain of the detection probe would have predictably increased the multiplexing capacity of the system taught by Zhang et al.
Regarding claim 52, Dunaway et al. teach detecting target sequences in native genomic DNA (Dunaway et al., paragraph 0325) or native RNA (i.e. naturally occurring RNA) without the need for cDNA conversion (Dunaway et al., paragraph 0331).
Regarding claim 54, Dunaway et al. teach optically detectable moieties linked to the reporter probes are fluorophores (Dunaway et al., paragraph 0198-0199).
Regarding claim 57, Dunaway et al. teach that the number of different species of reporter probes in the universal pool of reporter probes can be “at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or more” (Dunaway et al., paragraph 0168).
Regarding claim 58, Dunaway et al. teach differentiating between at least 512 distinct sequences (Dunaway et al., figure 3) (i.e. 50 or more barcodes)
Regarding claims 60-61, Dunaway et al. teach the overhang sequence is specific to the identity of the “target binding domain”. Therefore, the first and second overhang sequences can be the same or different.
Regarding claims 62-64, Dunaway et al. teach that the first and second domains may partially or fully overlap without regard to the particular molecular polarity of the detection probes (i.e. the order of the first and second domains along the target sequence are interchangeable) (Dunaway et al., paragraph 0272 and figure 14).
Regarding claims 65-66, Dunaway et al. teach multiple sequencing probes (i.e. detection probes) bind to overlapping target sequences (Dunaway et al., paragraph 0272) and that the sequencing probes can be removed (i.e. displaced) by partially overlapping sequencing probes comprising a toehold overhang (Dunaway et al., paragraph 0156).
Regarding claims 67-68, Dunaway et al. teach that each domain may comprise a unique subunit (Dunaway et al., figure 3).
Regarding claim 69, Zhang et al. teach the system comprises a ligase for circularizing the padlock probe (Zhang et al., page 12181, column 2, paragraph 2).
Regarding claims 70-71, Zhang et al. teach the system comprises phi29 DNA polymerase (i.e. a polymerase) for performing rolling circle amplification of a circularized padlock probe (Zhang et al., page 12181, column 2, paragraph 3).
Regarding claim 72, the systems disclosed by Dunaway for identifying particular target nucleic acid sequences are not limited to any particular target sequences. In fact, Dunaway teaches “a target nucleic acid can be any nucleic acid to which the sequencing probe… can hybridize… can be DNA or RNA… can be obtained from a biological sample…” (Dunaway et al., paragraph 0120). The teaching of Dunaway et al. that the combination of universal reporter probe fluorescent signals bound to a particular “sequencing probe” identifies a particular target sequence (Dunaway et al., figure 3) (e.g. a “barcode” or subunit thereof) is analogous to the combination of universal reporter probe fluorescent signals bound to a particular target sequence (e.g. a “barcode”) taught by Zhang et al. Furthermore, Zhang et al. teaches the target sequence is a known nucleic acid sequence comprising a combination of probe binding sites (i.e. is a barcode sequence on a padlock probe) (Zhang et al., figure 1).
Response to arguments
The response asserts that “Claims 51, 52, 54, 57, 58, and 60-71 are nonobvious over Zhang in view of Dunaway… because: (1) Zhang and Dunaway do not teach or suggest any padlock probe that comprises a nucleotide barcode sequence that identifies the padlock probe; (2) Zhang and Dunaway do not teach or suggest any padlock probe that comprises a nucleotide barcode sequence comprising partially overlapping first domain sequence and second domain sequence; (3) There is no reason or motivation for the wholesale replacement of Zhang’s ratiometric encoding with the “expanded encoding system” allegedly disclosed in Dunaway.”; (4) Dunaway is silent on padlock probes.
These arguments have been thoroughly reviewed and are not persuasive for the reasons which follow.
Zhang et al. does, in fact, teach padlock probes comprising barcode sequences that identify the padlock probe. It is well known in the art, for example, Goodwin et al., “Coming of age: ten years of next-generation sequencing technologies” Nature Reviews Genetics, 17, 333-351 (2016), cited here only in response to arguments, that a “Barcode” sequence is “a series of known bases added to a template molecule either through ligation or amplification. After sequencing, these barcodes can be used to identify which sample a particular read is derived from.” (Goodwin et al., page 345, left panel “Barcodes”). The known sequence of nucleotides comprising a number of “red” and “green” probe binding sites (i.e. a first and second domain sequence) in the padlock probes taught by Zhang et al. identify particular target sequences that are hybridized to particular padlock probes. Thus, the padlock probes taught by Zhang et al. comprise barcodes that identify the padlock probes. The further assertion presented in the response that the “neither the red probe site nor the green probe site sequence identifies a padlock probe from among the different padlock probes” in Zhang et al. is not persuasive for the same reasons cited above. In particular, Zhang et al. teaches the combination of multiple red and green probe sites identifies the padlock probe (i.e. the barcode comprises a first and second domain sequence…).
and (3): In response to applicant’s arguments that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Zhang et al. teach that the system for multiplex detection of nucleic acid barcode sequences comprising ratiometric detection of fluorescence signals may be expanded to a greater number of distinguishable fluorescence ratios (Zhang et al., page 12184, column 1, paragraph 2) by adding more barcode copies, shortening the length of the barcodes, and/or adding more distinct barcodes with their own fluorophore-labeled probes (Zhang et al., page 12185, column 2, paragraph 3). Additionally, Dunaway teaches that given a first or second domain sequence as short as 6 nucleotides and detection probes labeled with as few as two fluorophores allows for discrimination between 512 distinct target sequences based upon the combination of fluorescence signals read for the sequence (Dunaway et al., figure 3). As described above, the ordinary artisan would have been motivated to expand the number of distinguishable barcodes encoded by a set of padlock probes (Zhang et al., page 12185, column 2, paragraph 3) and would have predictably accomplished said expansion by the methods taught by Dunaway et al. comprising hundreds of distinct sequences in a nucleotide sequence to be determined (e.g. barcodes) using partially overlapping “sequencing probes” that are analogous to the “detection probes” of the present disclosure. The “sequencing probes” taught by Dunaway et al. comprise a “target binding domain” that hybridizes to a target sequence (i.e. is analogous to a first or second domain sequence) and a “barcode domain” that hybridizes to fluorescently-labeled reporter probes (i.e. is analogous to a first or second overhang sequence) (Dunaway et al., figure 3). Dunaway et al. further teach a series of sequencing probes (i.e. detection probes) sequentially characterize a target sequence (e.g. a barcode sequence) in a “sequencing cycle” (Dunaway et al., figure 14). Finally, Dunaway et al. teach that the “sequencing cycle” of figure 14 can be repeated any number of times and that the sequencing probes can bind to the target nucleic acid at a position that overlaps the position at which the first sequencing probe was bound during the first sequencing cycle (i.e. the sequencing probes overlap and are capable of initiating a strand displacement reaction) (Dunaway et al., paragraph 0272).
The response further asserts that Zhang requires that the “red probe site and the green probe site in the padlock probes do not overlap” because they are shown as non-overlapping in Zhang et al., figure 1 and “the skilled artisan would understand that this… allows the red… and green probes to hybridize to the RCP without interfering with each other” and displacing “red” or “green” probes “would render Zhang unsatisfactory for its intended purpose of using R/G ratios for multiplex encoding of padlock probes and target sequences”.
This argument is similarly not persuasive because the combination of Zhang et al. and Dunaway et al. does not directly hybridize the labeled “reporter” probes directly to the RCP, but rather to the intermediate primary “encoding” or “sequencing” probes. The combination of Zhang et al. and Dunaway et al. assigns combinations of “reporter” signals to particular “sequencing” probes that bind to particular sequences (i.e. domains) within the barcodes on the padlock probes. The measured fluorescence from each combination of species of reporter probes(s) bound to a particular “sequencing” probe that identifies the particular sequencing probe (and thus the sequence to which it is hybridized (e.g. a barcode “domain)) in the method/system taught by Zhang et al. is analogous to the “ratio” of fluorescence observed in the method/system taught by Zhang et al.
Relevant to arguments involving motivation to combine the references, the response further argues that “the cited disclosures in Dunaway certainly do not stand for the teaching that overlapping probes should be used in any and all detection systems that involve probe hybridization”. This argument is also not persuasive, at least because the systems disclosed by Dunaway for identifying particular target nucleic acid sequences are not limited to any particular target sequences. In fact, Dunaway teaches “a target nucleic acid can be any nucleic acid to which the sequencing probe… can hybridize… can be DNA or RNA… can be obtained form a biological sample…” (Dunaway et al., paragraph 0120).
The response even further argues that there is no motivation to substitute the ratiometric encoding of Zhang et al. with the “sequencing probe” and “reporter probe” encoding of Dunaway et al. This argument is likewise not persuasive. As described above, the combination of universal reporter probe fluorescent signals bound to a particular “sequencing probe” that identifies a particular target sequence (e.g. a “barcode” or subunit thereof) taught by Dunaway et al. is analogous to the combination of universal reporter probe fluorescent signals bound to a particular target sequence (e.g. a “barcode”) taught by Zhang et al.
The response argues that the combination of Zhang et al. and Dunaway et al. would render Zhang unsatisfactory for its intended purpose because Zhang et al. uses only two universal probes and Dunaway et al. used multiple universal “reporter” probes per target-specific “sequencing probe”, which “would be more expensive and complicated”. This is not persuasive. As described above, the teachings of Dunaway et al. comprising a primary “sequencing” probe specific to a particular known target sequence (e.g. a barcode or subunit thereof) and secondary “reporter” probes whose fluorescent signals in combination identify the particular sequencing probe predictably expands the encoding capacity and/or fluorescent signal of systems such as Zhang et al. comprising direct hybridization of reporter probes to target sequences. The assertion that the combination would be more complicated or expensive would not have dissuaded the ordinary artisan from pursuing the obvious benefits of increased multiplexing relative to the methods of Zhang et al. alone discussed above.
The assertion that Dunaway does not teach padlock probes is not persuasive because Dunaway is not cited for a teaching of padlock probes in the rejection of record. Dunaway teaches (as described above) systems and methods for determining a large number of particular sequences (e.g. barcodes, domain sequences, etc.) comprising primary probes that are analogous to the claimed partially overlapping “detection probes” and secondary probes that are optically labeled and are analogous to the claimed “reporter probes”. The assertion that Dunaway alone does not teach this system is applied to identifying padlock probes amounts to a piecemeal analysis of the references cited in combination.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Claim 53 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. in view of Dunaway et al., as applied to claims 51-52, 54, 57-58, and 60-71 above, and further in view of Schweitzer et al., “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection” PNAS. August 29, 2000, 97, 18, 10113-10119.
Regarding claim 53, Zhang in view of Dunaway et al. teach a system for detecting a nucleic acid barcode sequence comprising a barcoded padlock probe that hybridizes to a target sequence, a panel of detection probes that hybridize to the barcode(s) in the rolling circle amplification product produced from the ligated padlock probe, and universal reporter probes that hybridize to an overhang sequence on the detection probes, wherein the detection probes are capable of initiating strand displacement reactions to displace overlapping detection probes from the barcode sequence on the rolling circle amplification product (see 103 rejection above).
Zhang et al. in view of Dunaway et al. do not teach that the target nucleic acid is linked to an antibody.
However, Schweitzer et al. teach systems for ultrasensitive detection of antigens wherein a nucleic acid molecule (i.e. the target) is linked to an antibody. Schweitzer teaches that a DNA circle (i.e. a ligated padlock probe) hybridizes to the target nucleic acid (i.e. the nucleic acid linked to the antibody) and serves as a template for rolling circle amplification. Schweitzer teaches directly detecting the rolling circle product with a fluorescent oligonucleotide probe (Schweitzer, figure 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the system for detection of native nucleic acid molecules with a barcoded padlock probe, a plurality of detection probes, and a pool of universal reporter probes, taught by Zhang et al. in view of Dunaway et al. to detect the presence of a tag sequence linked to an antibody, as taught in the system taught by Schweitzer et al. The ordinary artisan would have been motivated to detect the labeled antibodies of Schweitzer et al. in this way by the teachings of Zhang et al. and Dunaway et al. that detection of multipartite barcoded padlock probes can be further multiplexed using a combination of detector and labeled reporter probes. The ordinary artisan would therefore have predicted that the increased multiplexing capacity of the nucleic acid detection system taught by Zhang et al. in view of Dunaway et al. would readily have allowed for multiplexing of nucleic acid-tagged antibodies in the system taught by Schweitzer et al.
Response to arguments
The response asserts that “Schweitzer does not cure the deficiencies of Zhang and Dunaway”. This argument has been considered and is not persuasive for at least all of the reasons discussed above. These further assertions fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Claim 59 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. in view of Dunaway et al., as applied to claims 51-52, 54, 57-58, and 60-71 above, and further in view of Chen et al., “Efficient in situ barcode sequencing using padlock probe-based BaristaSeq”. Nucleic Acids Research, 2017, Vol. 46, No. 4 e22.
Regarding claim 59, Zhang in view of Dunaway et al. teach a system for detecting a nucleic acid barcode sequence comprising a barcoded padlock probe that hybridizes to a target sequence, a panel of detection probes that hybridize to the barcode(s) in the rolling circle amplification product produced from the ligated padlock probe, and universal reporter probes that hybridize to an overhang sequence on the detection probes, wherein the detection probes are capable of initiating strand displacement reactions to displace overlapping detection probes from the barcode sequence on the rolling circle amplification product (see 103 rejection above).
Zhang et al. in view of Dunaway et al. do not teach that the system comprises a cell or a tissue sample on a solid substrate comprising the target nucleic acid molecule.
However, Chen et al. teach a system for in situ (i.e. in a cell or tissue sample on a solid substrate) barcode sequencing of padlock probes (Chen et al., abstract).
Chen et al. teach that in situ sequencing of padlock probe barcodes has predictable advantages over “conventional approaches to reading out cellular barcodes” including “achieving both high spatial resolution and high throughput” whereas “bulk sequencing achieves high throughput but sacrifices spatial resolution, whereas manual cell picking is low throughput” (Chen et al., page 1, column 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the system taught by Zhang et al. in view of Dunaway et al. for high throughput, in situ detection (i.e. in a cell or tissue sample on a solid substrate) of the target sequences corresponding to the barcoded padlock probes of the system of Zhang et al. in view of Dunaway et al.
The ordinary artisan would have been motivated by the teaching of Chen et al. that in situ sequencing of padlock barcodes allows for high throughput and high spatial resolution in detecting specific nucleic acids species within a whole cell or tissue (Chen et al., page 1, column 1). The ordinary artisan would likewise have had a reasonable expectation that the padlock, detector, and reporter probes taught by Zhang et al. in view of Dunaway et al., would have predictably improved the throughput of the system taught by Chen et al. comprising direct, in situ sequencing of padlock barcode sequences by increasing the capacity for multiplexing described by Dunaway et al. (i.e. at least hundreds of uniquely labeled detector probes).
Response to arguments
The response asserts that “Chen does not cure the deficiencies of Zhang and Dunaway”. This argument has been considered and is not persuasive for at least all of the reasons discussed above. These further assertions fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Claims 51-54 and 57-72 are rejected under 35 U.S.C. 103 as being unpatentable over Peng, “Sequential Color Display for Highly Multiplexed In Situ Single-Molecule Detection” Dissertation, Stanford University (2017) (cited as NPL No. 004 on IDS filed on March 23, 2026) in view of Dunaway et al., US 2018/0142286 A1, published May 24, 2018.
This is a new grounds of rejection necessitated by the IDS filed on March 23, 2026.
Regarding claim 1, Peng teaches a system for detecting a target nucleic acid sequence comprising: (a) padlock probes comprising known “detection sequences” (i.e. a barcode) that hybridizes to the target sequence (b) a universal pool of reporter probes comprising at least 2 species of reporter probes each having an optically detectable moiety (c-d) a first and second detection probe comprising a sequence that hybridizes to the padlock barcode and an overhang sequence comprising reporter probe binding sites (Peng, figure 3.2, reproduced below for convenience).
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Peng does not appear to teach that the detection probes partially overlap on the RCP comprising the known padlock barcode sequence.
However, Dunaway et al. teach probe sets comprising primary “sequencing probes” that are analogous to the “detection probes” of the present disclosure. The “sequencing probes” taught by Dunaway et al. comprise a “target binding domain” that hybridizes to a target sequence (i.e. is analogous to a first or second domain sequence) and a “barcode domain” that hybridizes to fluorescently-labeled reporter probes (i.e. is analogous to a first or second overhang sequence) (Dunaway et al., figure 3).
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Dunaway et al. further teach that a series of sequencing probes (i.e. detection probes) can be used to sequentially characterize a target sequence in a “sequencing cycle” (Dunaway et al., figure 14). Finally, Dunaway et al. teach that the “sequencing cycle” of figure 14 can be repeated any number of times and that the sequencing probes can bind to the target nucleic acid at a position that overlaps the position at which the first sequencing probe was bound during the first sequencing cycle (i.e. the sequencing probes overlap and are capable of initiating a strand displacement reaction) (Dunaway et al., paragraph 0272).
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the padlock probe system for detection of padlock probes taught by Peng with the teachings of Dunaway et al. comprising “sequencing probes” (i.e. detection probes) that hybridize to a target sequence that can overlap between two or more sequencing probes and fluorescently labeled, universal “reporter probes” that hybridize to an overhang domain of the “sequencing probes”. The ordinary artisan would have been motivated to use the partially overlapping detection probes and reporter probes taught by Dunaway et al. to detect the multiplexed padlock barcodes taught by Peng because of the suggestion of Dunaway et al. that probes comprising toe-hold sequences accelerate the rate of exchange of oligonucleotides hybridized adjacent to a toehold sequence (Dunaway et al., paragraph 0156). Further, Dunaway et al. teaches that given a first or second domain sequence as short as 6 nucleotides and detection probes labeled with as few as two fluorophores allows for discrimination between 512 distinct target sequences (Dunaway et al., figure 3). Both Peng and Dunaway et al. teach nucleic acid systems for detecting specific target systems using combinations of fluorescent signals localized to the target nucleic acid. Therefore, the ordinary artisan would have had a reasonable expectation that the partially overlapping “sequencing probes” (i.e. detection probes) comprising toe-hold sequences, taught by Dunaway et al. would have accelerated the rate of exchange of “detection probes” in the methods and systems taught by Peng.
Regarding claim 52, Regarding claim 52, Dunaway et al. teach detecting target sequences in native genomic DNA (Dunaway et al., paragraph 0325) or native RNA (i.e. naturally occurring RNA) without the need for cDNA conversion (Dunaway et al., paragraph 0331).
Regarding claim 53, Peng teach the target nucleic acid molecule is linked to an antibody (Peng, figure 3.1, reproduced below for convenience)
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Regarding claim 54, Dunaway et al. teach optically detectable moieties linked to the reporter probes are fluorophores (Dunaway et al., paragraph 0198-0199).
Regarding claim 57, Dunaway et al. teach that the number of different species of reporter probes in the universal pool of reporter probes can be “at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or more” (Dunaway et al., paragraph 0168).
Regarding claim 58, Dunaway et al. teach differentiating between at least 512 distinct sequences (Dunaway et al., figure 3) (i.e. 50 or more barcodes)
Regarding claim 59, Peng teaches the system further comprises a biological sample comprising the target nucleic acid wherein the biological sample is cells or tissues fixed by formaldehyde on a solid surface (Peng, page 32, paragraph 2).
Regarding claims 60-61, Dunaway et al. teach the overhang sequence is specific to the identity of the “target binding domain”. Therefore, the first and second overhang sequences can be the same or different.
Regarding claims 62-64, Dunaway et al. teach that the first and second domains may partially or fully overlap without regard to the particular molecular polarity of the detection probes (i.e. the order of the first and second domains along the target sequence are interchangeable) (Dunaway et al., paragraph 0272 and figure 14).
Regarding claims 65-66, Dunaway et al. teach multiple sequencing probes (i.e. detection probes) bind to overlapping target sequences (Dunaway et al., paragraph 0272) and that the sequencing probes can be removed (i.e. displaced) by partially overlapping sequencing probes comprising a toehold overhang (Dunaway et al., paragraph 0156).
Regarding claims 67-68, Dunaway et al. teach that each domain may comprise a unique subunit (Dunaway et al., figure 3).
Regarding claim 69, Peng teach the system comprises ampligase (i.e. a ligase) for ligation of the padlock probe to generate a circularized padlock probe (Peng, page 62-63, bridging paragraph).
Regarding claims 70-71, Peng. teach the system comprises phi29 DNA polymerase (i.e. a polymerase) for performing rolling circle amplification of a circularized padlock probe (Peng, page 63, paragraph 2).
Regarding claim 72, the systems disclosed by Peng comprise detection of nucleic-acid tagged proteins (e.g. antibodies) (i.e. the nucleic-acid tags are barcodes) wherein the padlock probe binds to a target sequence on the nucleic acid tag (Peng, figure 3.1).
Conclusion
No claim is allowed.
Applicant's submission of an information disclosure statement under 37 CFR 1.97(c) with the timing fee set forth in 37 CFR 1.17(p) on March 23, 2026 prompted the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 609.04(b). 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 ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at 5712723157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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