DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 19 November 2025 has been entered.
Applicant is notified that the examiner for this action has changed.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-73, 75, 78, and 92 were previously cancelled.
Claims 74, 82, 89, and 93 are amended.
Claims 74, 76-77, 79-91, and 93-96 are being examined.
Claim Interpretation
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 § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 90 and 91 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Based on the interpretation of claim 74, lines 9-10 described in the Claim Interpretation section above, claim 74 requires that the modification of the probe by attachment of nucleotides based on the first oligonucleotides as a template occur after the probe is hybridized to the sample. Claims 90 and 91 fail to further limit the subject matter of claim 74 upon which each claim depends because the above-described limitation of claim 74 requires that the attaching step be performed after contacting the sample comprising the target nucleic acid with the probe and the first oligonucleotide and after the probe is hybridized to the target nucleic acid. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
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, 79-91, 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. (WO 2014/163886, published 9 October 2014), herein Church, and in view of Thom (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 overhang 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 nucleotides 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 Daugharthy’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 Daugharthy 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 the 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 been 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, line 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, and octadiynyl dU, at 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 or 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, line 65-68; col 12, line 34-36; col 25, line 7-11; Figure 1).
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 (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 90, Thom teaches a method wherein the attaching step is performed after contacting the sample comprising the target nucleic acid with the probe and the first oligonucleotide ([0107, 0112, 0113]; Figure 9A-B).
Regarding claim 91, Thom teaches a method wherein the attaching step is performed after the probe is hybridized to the target nucleic acid ([0107, 0112, 0113]; Figure 9A-B).
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 Remarks
Applicant’s arguments, see pages 6-10 of remarks, filed 19 November 2025, with respect to the rejections of claims 74, 81, 82, 84-91, 95, and 96 under Daugharthy et al. (US 10,844,426), herein Daugharthy, in view of Frisen et al. (WO 2018/091676), herein Frisen; claims 79 and 80 under Daugharthy in view of Frisen and further in view of Cai et al. (WO 2018/026873), herein Cai; claims 93 and 94 under Daugharthy in view of Cai in further view of Frisen; claims 76, 77, and 83 under Daugharthy in view of Frisen and further in view of Chen et al. (US 10,844,426), herein Chen, have been fully considered and are persuasive in part. Applicant’s argument that 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” and that Frisen, Cai, and Chen fail to cure this deficiency is persuasive. Therefore, the rejections have been withdrawn. However, Applicant has not disputed that Daugharthy teaches the limitation: “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 overhang hybridizes to the first oligonucleotide.” Upon further consideration, two new grounds of rejection are made. First, in view of the updated interpretation of claim 74 lines 9-10 (see Claim Interpretation), claims 90 and 91 are rejected under 35 U.S.C. 112(d) as being of improper dependent form (see Claim Rejections - 35 USC § 112(d)). Second, claims 74, 76-77, 79-91, and 93-96 are rejected under 35 U.S.C. 103 as being unpatentable over the undisputed teaching of Daugharthy in view of the newly cited teaching of Church and the newly cited teaching of Thom as evidenced by the undisputed teaching of Nejedly (see Claim Rejections - 35 USC § 103).
Conclusion
All claims are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jeffrey Lawrence Bellah whose telephone number is (571)272-1024. The examiner can normally be reached M-Th, 7:30-5 ET.
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, Anne Gussow can be reached at (571)272-6047. 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.
/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683