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 .
Withdrawn Rejections
All rejections of claims 90 and 91 are withdrawn in view of claims 90 and 91 being canceled in the amendment to the Claims filed 21 April 2026.
Claim Status
Claims 1-73, 75, 78, and 90-92 are canceled.
Claims 74, 76-77, 79-89, and 93-96 are previously presented and under examination.
Claim Interpretation
Maintained
In claim 74 lines 9-10 and claim 93 lines 11-12, these claims recite “thereby modifying the probe hybridized to the target nucleic acid in the sample”. Though this is a statement of the outcome of step (b), the use of the past tense “hybridized” instead of a descriptor of ability (such as “capable of hybridizing to”) is interpreted as adding further limitation to the step such that the modification of a probe by extension using a polymerase has to occur to a probe that is hybridized to a target nucleic acid in the sample.
Claim Rejections - 35 USC § 103
Maintained
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 74, 76-77, 79-89, and 93-96 are rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. (US 10,844,426, US PAT 121 on IDS dated 6 May 2022), herein Daugharthy, in view of Church et al. (provided in previous office action)(WO 2014/163886, published 9 October 2014), herein Church, and in view of Thom (provided in previous office action)(WO 2020/227543, published 12 November 2020) as evidenced by Nejedly et al. (provided in previous office action)(“Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA”, Biochim Biophys Acta, 1517(3), pages 365-375 (2001)).
Regarding claim 74, Daugharthy teaches a method of identifying a target nucleic acid through the use of a probe that hybridizes to the target nucleic acid (Figure 6A), with two overhangs that do not bind to the target nucleic acid (Figure 6A mainstreet and backstreet), where the second overhang binds to an oligonucleotide (Figure 6A mainstreet binding to secondary; col 2, lines 29-32), and where probes are modified to incorporate a functional moiety by introducing nucleotides modified with a functional group into the probe (col 19, lines 58-66), reading on the limitations: “contacting a probe, a first oligonucleotide, and a sample comprising a target nucleic acid in any suitable order, wherein: the probe comprises (i) a hybridization region that hybridizes to the target nucleic acid in the sample (ii) a first overhang, and (iii) a second overhang, wherein the first and second overhangs do not hybridize to the target nucleic acid, and the second overhand hybridizes to the first oligonucleotide and b) to attach one or more modified nucleotides to [the probe]”. Daugharthy also teaches the method further comprising crosslinking the one or more modified nucleotides to the sample, a substrate, and/or a matrix (col 19, lines 60-62) and detecting a signal indicative of the probe hybridized to the target nucleic acid at a location in the sample (abstract; Figure 4; col 9, lines 34-49).
However, Daugharthy only teaches that the modified nucelotides are added “during amplification” (col 19, line 66), which is part of making the probes (col 14, lines 12 and 22-30), so Daugharthy does not teach “extending the probe using a polymerase to attach one or more modified nucleotides to the second overhang using the first oligonucleotide as a template, thereby modifying the probe hybridized to the target nucleic acid in the sample”, which requires use of a polymerase and the first oligonucleotide as a template and for the extension to occur after the probe is hybridized to the target (see Claim Interpretation section above). These deficiencies are made up for in the combined teachings of Thom and Church.
Thom teaches a method of modifying target-specific probes that bind to a nucleic acid target through hybridization ([0016, 0058, 0110], Figure 9A, legend for Figure 9 is in Figure 8A) by contacting the sample and oligonucleotides with a polymerase ([0005, 0022, 0124]), hybridizing an overhang of a first oligonucleotide probe (equivalent to the instant probe) to a second oligonucleotide probe (equivalent to the instant first oligonucleotide), and extending the first oligonucleotide probe along the second oligonucleotide probe to form a first extension product (equivalent to the instant modified probe)([0107, 0112, 0113, 0128], Figure 9B).
Though Thom teaches using a polymerase to attach nucleotides, Thom does not teach using a polymerase to attach modified nucleotides. This deficiency is made up for in the teachings of Church.
Church teaches attaching modified nucleotides, which are capable of crosslinking, by an amplification reaction (page 6, lines 3-6) such as PCR (polymerase chain reaction)(page 10, lines 27-28), as well as teaching examples using both MMuLV reverse transcriptase (a polymerase) and phi29 DNA polymerase to attach aminoallyl modified nucleotides (page 15, line 23 – page 16 line; Figure 10). Therefore, the combination of Thom and Church reads on the limitation: “extending the probe using a polymerase to attach one or more modified nucleotides to the second overhang using the first oligonucleotide as a template, thereby modifying the probe hybridized to the target nucleic acid in the sample”.
In view of Duagharthy’s teaching that incorporating modified nucleotides into the probe is advantageous for attaching the probe to a matrix material and that Church (WO 2014/163886) is literature describing exemplary methods of doing so (Daugharthy, col 19, lines 47-57), one of ordinary skill in the art would be motivated to combine the Daugharthy and Church references. One of ordinary skill in the art would have a reasonable expectation of success in this combination because using a polymerase for incorporation of nucleotides is standard in the art, as evidenced by Church using multiple polymerases for this purpose. Therefore, the combination of Daugharthy with Church would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Because Church describes the ability to use polymerase to incorporate modified nucleotides, one of ordinary skill in the art would understand that other methods that use polymerase to incorporate nucleotides could be substituted for the amplification described in Daugharthy (col 19, line 66). Thom teaches one such alternative method, wherein the probe being extended by a polymerase incorporating additional nucleotides is already hybridized to a target and is extended based on an oligonucleotide other than the probe or target. There would be a reasonable expectation of success, because it would only involve swapping similar steps that serve the same purpose of incorporating nucleotides into a probe with a polymerase and because Thom demonstrates that the extension of the probe can be achieved in situ after the probe is bound to the target. Thus, the combination of Duagharthy and Church with Thom would have been an obvious simple substitution (MPEP §2143 I. B.) to one of ordinary skill in the art before the effective filing date of the claimed invention. Because the combination of Daugharthy, Church, and Thom would have been obvious, the invention as a whole was obvious before the effective filing date.
It would have been prima facie obvious before the effective filing date of the claimed invention for a person having ordinary skill in the art to have used the method of extending a probe that is bound to a sample using a polymerase and a separate template oligonucleotide as taught by Thom and the method of using a polymerase to attach modified nucleotides as taught by Church with the method of detecting a probe hybridized to a target nucleic acid at a location in a sample by using a probe containing modified nucleotides and with two overhangs that hybridizes to a target nucleic acid and crosslinking the modified nucleotides of the probe to a matrix as taught by Daugharthy. Each element of the claimed invention is taught in the prior art, as discussed above, with the only difference being the lack of the specific combination of the elements in a single prior art reference. In the combination of Daugharthy, Thom, and Church, each element merely performs the same function as it does separately: the probe binds a target through hybridization, has modified nucleotides crosslinked to a matrix, and is detected the same as it does in Daugharthy; an overhang hybridizes to a separate oligonucleotide and is extended by a polymerase using the separate oligonucleotide as a template as it does in Thom; and a polymerase attaches modified nucleotides in an extension reaction as it does in Church. Because each element is only performing the same function as it is known to perform in the art, one of ordinary skill in the art would have recognized that the results of the combination of the methods of Daugharthy, Thom, and Church were predictable and would have a reasonable expectation of success. “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. KSR, 550 U.S. at 416, 82 USPQ2d at 1395” (MPEP §2143 I. A.). Thus, the combination of Daugharthy and Church with Thom would have been an obvious combination of prior art elements to one of ordinary skill in the art before the effective filing date of the claimed invention. Because the combination of Daugharthy, Church, and Thom would have bene obvious, the invention as a whole was obvious before the effective filing date.
Regarding claim 76, Thom teaches a method wherein the first oligonucleotide is blocked at the 3’ from extension by a 3’ modification ([0038, 0056]; Figure 11).
Regarding claim 77, Thom teaches a method wherein the first oligonucleotide comprises a 3’ ddC (3’ dideoxy-C)([0056, 0095]).
Regarding claim 79, Thom teaches a method wherein the method further comprises contacting the sample with a second oligonucleotide, wherein the second oligonucleotide hybridizes to the second overhang of the modified probe ([0113, 0124]; Figure 9C).
Regarding claim 80, Thom teaches a method wherein the method further comprises extending the modified probe with a polymerase to attach one or more nucleotides to the second overhang of the modified probe using the second oligonucleotide as a template (Thom [0005, 0022, 0113, 0124]; Figure 9C), and Church teaches the attachment of modified nucleotides by polymerase (Church page 10, lines 27-28; page 15, lines 23 – page 16 line; Figure 10), thereby further modifying the probe hybridized to the target nucleic acid in the sample.
Regarding claim 81, both Daugharthy and Church teach methods wherein the one or more modified nucleotides comprise one or more cross-linkable nucleotides and/or wherein the one or more modified nucleotides comprise a halogenated base, an azide-modified base, an octadiynyl dU, a thiol-modified base, a biotin-modified base, or a combination thereof (Daugharthy, col 19, line 60 – col 20 line 3; Church, page 6, lines 2-8).
Regarding claim 82, both Daugharthy and Church teach methods wherein the one or more modified nucleotides are crosslinked to a matrix (Daugharthy, col 19, lines 58-60; Church, page 6, lines 1-2).
Regarding claim 83, Church teaches a method wherein the one ore more modified nucleotides comprise at least one nucleotide that is internal after incorporation (Figure 10).
Regarding claim 84, Daugharthy teaches a method wherein the first overhang comprises one or more barcode sequences (col 1, lines 55-57; col 4, lines 65-68; col 12, line 34-36; col 25, line 7-11; Figure 11).
Regarding claim 85, Daugharthy teaches a method wherein the first overhang comprises one or more landing sequences capable of hybridizing to one or more secondary probes (col 2, lines 29-32; col 10, lines 1-12; Figure 6A).
Regarding claim 86, Daugharthy teaches a method wherein the one or more secondary probes are detectably labeled (col 2, lines 29-32; col 10, lines 1-12; Figure 6A).
Regarding claim 87, Daugharthy, Thom, and Church teach methods wherein the sample is a tissue sample (Daugharthy, col 21, line 54-55; Thom [0106]; Church, page 2, line 9-11).
Regarding claim 88, Daugharthy and Thom teach methods wherein the method further comprises analyzing localization of the target nucleic acid in the sample (Daugharthy, Figures 4 and 5, col 24, lines 13-16; Thom [0012]).
Regarding claim 89, Daugharthy teaches a method wherein the signal indicative of the probe hybridized to the target nucleic acid in the sample corresponds to the one or more barcode sequences (Figures 4-6; col 9, lines 34-39).
Regarding claim 93, Thom teaches a method of modifying a probe wherein the target nucleic acid is an endogenous RNA ([0123]) and the remaining limitations are taught by the combination of Daugharthy, Thom, and Church as described above for claim 74.
Regarding claim 94, Daugharthy teaches a method wherein the first overhang comprises a barcode which the secondary probe hybridizes to (col 10, lines 1-12; Figures 1 and 6A).
Regarding claim 95, Daugharthy teaches a method wherein the nucleotides are adenine, cytosine, guanine, and thymine (col 22, lines 28-30), which are photo-crosslinkable nucleotides (as evidenced by Nejedly et al., abstract; Introduction paragraph 2; Figure 2).
Regarding claim 96, Daugharthy teaches a method wherein the nucleotides are adenine, cytosine, guanine, and thymine (col 22, lines 28-30), which are UV-crosslinkable nucleotides (as evidenced by Nejedly et al., abstract; Introduction paragraph 2; Figure 2).
Response to Arguments
Applicant’s arguments filed 21 April 2026 have been fully considered but they are not persuasive. In the arguments filed 21 April 2026, Applicant argues that:
Daugharthy teaches probe modification during probe synthesis before the probe is contacted with the sample, in contrast to the claimed method that modifies the probe that is already hybridized to the target nucleic acid in the sample (Remarks page 6 last paragraph).
The cited references do not provide any motivation for one skilled in the art to modify Daugharthy to extend an overhang of a probe using a polymerase to attach one or more modified nucleotides to the overhang using an oligonucleotide as a template to modify the probe hybridized to the target nucleic acid in the sample because Daugharthy teaches a probe modified during probe synthesis and has no problem that would be solved by post-hybridization, in-sample modification (Remarks page 7 second and third paragraphs).
Thom teaches probes functionalized with immobilization moieties added during probe synthesis before contacting the probes with a sample (Remarks page 8 second paragraph).
Thom does not describe any extension reactions performed to modify a probe with modified nucleotides for crosslinking to a sample, substrate, and/or matrix, and Thom teaches the immobilization of probes to the sample prior to and independent of the extension depicted in FIG. 9B (Remarks page 9 first paragraph).
Based on arguments (1)¸(3), and (4), even if the skilled artisan combined Daugharthy and Thom (which argument (2) argues there is no teaching, suggestion, or motivation to do so), one skilled in the art would choose the immobilization method using probes modified during synthesis prior to contacting a probe with a sample rather than modifying the probe hybridized to the target nucleic acid in the sample (Remarks page 9 second paragraph).
Based on the prior arguments, Thom does not teach using extension to modify a probe hybridized to the target nucleic acid in the sample or any motivation to do so, and Church also does not cure this deficiency (Remarks page 10 first paragraph).
Regarding argument (1), this is an accurate statement of a difference between the teachings of Daugharthy and the claimed method, but on its own is not persuasive because those differences are provided in the teachings of Church and Thom and because 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).
Regarding argument (2), MPEP §2143 states:
The Supreme Court in KSR Int’L Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1895-97 (2007) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper “functional approach” to the determination of obviousness as laid down in Graham. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit. In Ball Aerosol v. Ltd. Brands, 555 F.3d 984, 89 USPQ2d 1870 (Fed. Cir. 2009), the Federal Circuit offered additional instruction as to the need for an explicit analysis. The Federal Circuit explained that the Supreme Court’s requirement for an explicit analysis does not require record evidence of an explicit teaching of a motivation to combine in the prior art. (emphasis added)
Therefore, while a rationale to support a conclusion of prima facie obviousness is required, an explicit teaching, suggestion, or motivation to combine in the prior art is not required for the invention to be prima facie obvious.
In the Office Action mailed 26 January 2026, rationale to combine the teachings of Daugharthy, Church, and Thom are provided on page 6, beginning with the paragraph that starts “In view of Daugharthy’s teaching…”, through page 7. It is noted by the Examiner that Applicant does not dispute the rationale to combine the teachings of Daugharthy with the teachings of Church provided in the paragraph beginning with “In view of Daugharthy’s teaching…”. The paragraph beginning with “Because Church describes…” provides a rationale to combine the teaches of Thom with the combined teachings of Daugharthy and Church based on substituting one known element (the incorporation of nucleotides into a probe by the extension of an oligonucleotide probe hybridized to a target nucleic acid using a second oligonucleotide as a template, as taught by Thom) for another (the incorporation of nucleotides into a probe by amplification during probe synthesis, as taught by Daugharthy) to obtain the predictable result of incorporating desired nucleotides into a probe. As discussed in MPEP §2143 I. B., the simple substitution of one known element for another to obtain predictable results in one such rationale for supporting a conclusion of prima facie obviousness. Examiner notes that Applicant does not provide any arguments for why the described substitution of one method of incorporating nucleotides into a probe for another method of incorporating nucleotides into a probe would not yield the predictable result of incorporating desired nucleotides into a probe. While the two above discussed paragraphs provide a rationale to combine the teachings of Daugharthy, Church, and Thom, supporting Examiner’s conclusion of prima facie obviousness, in the Office Action mailed 26 January 2026 an additional rationale to combine the teachings of Daugharthy, Church, and Thom by combining prior art elements according to known methods to yield predictable results (see MPEP §2143 I. A.) is provided in the paragraph beginning with “It would have been prima facie obvious…”. Examiner notes that Applicant does not provide any arguments directly rebutting this second rationale to combine the references either.
Though Applicant argues in argument (2) that there is no motivation for one skilled in the art to modify Daugharthy to modify a probe hybridized to the target nucleic acid in the sample as claimed because there is no problem in Daugharthy that would be solved by post-hybridization functionalization, this argument is not persuasive because an explicit motivation is not required and because Applicant does not provide any arguments rebutting the rationales to combine provided in the Office Action mailed 26 January 2026.
Regarding argument (3), while it is accurately argued that Thom teaches oligonucleotides that are functionalized by adding reactive groups during synthesis, this argument is not persuasive because the teaching of Thom used in the 35 U.S.C. 103 rejections of claims 74 and 93 (and claims 76-77, 79-89, and 94-96 based on their dependency on either claim 74 or 93) is the teaching that an oligonucleotide probe hybridized to a target nucleic acid can be modified (in the sense of being changed) by incorporating nucleotides through an extension reaction using a separate oligonucleotide as a template. These teachings are not mutually exclusive, as the functionalization of a probe by the incorporation of modified nucleotides with reactive groups during probe synthesis does not prevent the probe from being further extended by a polymerase in an extension reaction. Furthermore, the teaching of Thom in Applicant’s argument does not teach away from the claimed invention, instead it just provides one exemplary way that oligonucleotides may be functionalized (teachings of alternatives do not constitute teaching away, see MPEP §2145 X. D. 1. “Furthermore, “the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of those alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004)”). Therefore, the argument that Thom teaches oligonucleotides that are functionalized by adding reactive groups during synthesis does not persuade Examiner that Thom does not teach the teaching relied upon in the 35 U.S.C. 103 rejections of claims 74 and 93 (and claims 76-77, 79-89, and 94-96 based on their dependency on either claim 74 or 93).
Regarding argument (4), while it is accurately argued that Thom does not teach the incorporation of modified nucleotides into a probe that is hybridized to a sample, this argument on its own is not persuasive because, while Thom provides the incorporation of nucleotides into a probe that is hybridized to a sample (the extension reaction), the teaching of the incorporation of modified nucleotides by polymerase is provided in the teaching of Church and because 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). Regarding Applicant’s argument that Thom teaches the immobilization of probes to the sample prior to and independent of the extension depicted in FIG. 9B, Examiner points out that paragraph [0157] of Thom recites “FIG. 9A shows immobilization (binding) of probes to the sample” (emphasis added). As immobilization is equated to binding in the context of FIG. 9 and Thom teaches that probes may bind a nucleic acid target through hybridization (“target specific probes (e.g., oligonucleotides) that specifically bind to one or more targets in the sample. For example, probes may bind a nucleic acid target (e.g., RNA) through hybridization” [0016]), one of ordinary skill in the art would understand Thom to be teaching that the extension reaction can be done after binding (via hybridization) of the probe to the target nucleic acid. This teaching of Thom, the modification of a probe hybridized to a target nucleic acid by extending the probe to incorporate nucleotides based on a separate template oligonucleotide, is the teaching of Thom utilized in the combination of Daugharthy, Church, and Thom in the 35 U.S.C. 103 rejections of claims 74 and 93 (and claims 76-77, 79-89, and 94-96 based on their dependency on either claim 74 or 93). Therefore, argument (4) does not persuade Examiner that Thom does not teach the teaching relied upon in the 35 U.S.C. 103 rejections of claims 74 and 93 (and claims 76-77, 79-89, and 94-96 based on their dependency on either claim 74 or 93).
Regarding argument (5), this argument is not persuasive. Firstly, as discussed with regard to argument (2), there is rationale to combine the teachings of Daugharthy, Church, and Thom even in the absence of an explicit teaching, suggestion, or motivation. Secondly, the assertion that one skilled in the art would choose an immobilization method using probes modified during synthesis instead of the claimed method is a mere allegation of what one skilled in the art may find preferable and is not sufficient to constitute either a reference teaching away from the combination or evidence of accepted wisdom that the claim proceeds contrary to. Therefore, this argument is not persuasive.
Regarding argument (6), Thom teaches using an extension reaction to modify a probe hybridized to a target nucleic acid in a sample by incorporating nucleotides (see discussion of argument (4) above; see also 35 U.S.C. 103 rejections of claims 74 and 93 regarding Thom above), leaving the sole deficiency in the combination of Daugharthy and Thom being the lack of teaching the incorporation of modified nucleotides by a polymerase. Church’s teaching of incorporating modified nucleotides into an extension product of a probe/primer bound to a nucleic acid target in situ by a polymerase (such as in Figure 10 where the Phi29 polymerase incorporates aminoallyl dUTP into the extension product for obtaining spatial information) does cure the deficiency of the combination of Daugharthy and Thom. Therefore, argument (6) is not persuasive.
As none of arguments (1)--(6) are persuasive, the rejection of claims 74, 76-77, 79-89, and 93-96 under 35 U.S.C. 103 as being unpatentable over Daugharthy in view of Church and Thom is maintained.
Conclusion
Claims 74, 76-77, 79-89, and 93-96 are rejected.
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.
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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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