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 17/881,086 filed on 08/04/2022 claims the benefit of provisional U.S. Patent Application No. 63/229,313, filed on 08/04/2021.
The priority date of claims 1-20 is determined to be 08/04/2021, the filing date of provisional U.S. Patent Application No. 63/229,313.
Status of Claims
Applicant’s amendments to claims filed 08/14/2025 in response to the Non-Final Rejection mailed 05/20/2025are acknowledged.
Claims 3 and 11 are amended.
New claim 21 is acknowledged.
Claims 1-21 are pending and under examination.
Response to Remarks filed 08/14/2025
The amendments and arguments presented in the papers filed 08/14/2025 ("Remarks”) have been thoroughly considered. The issues raised in the Office action dated 05/20/2025 listed below have been reconsidered as indicated.
a) The objections to the specification regarding the use of trade names or marks are withdrawn in view of the amendments to the specification.
b) The 35 USC 112(b) indefiniteness rejections of claims 3 and 11 have been withdrawn in view of the amendments to claims 3 and 11.
New and modified grounds of rejection necessitated by amendment are detailed below and this action is made FINAL.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-8, 10-20, and 21 remain/are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Frenz et al. (WO2020123316A2, on IDS dated 04/21/2023. Filed 12/06/2019 and published 06/18/2020).
The rejection of claim 21 is necessitated by claim amendments filed on 08/14/2025 including new claim 21.
Regarding claim 1, Frenz teaches methods for determining a location of a biological analyte in a biological sample using capture probes. Frenz teaches the use of capture probes (i.e. hydrogel-binding probes) that include capture domains capable of binding to an analyte (p 387, lines 7-9). Frenz further teaches that capture probes can include synthesized oligonucleotides (p 106, line 13) that can further include biotinylated oligonucleotides in addition to sequences used to capture the analyte (i.e. capture domain) (p 252, lines29-30). Frenz also teaches the use of acrydite moieties (i.e. a hydrogel-binding moiety) that can be modified to form chemical bonds with a capture probe (p 160, lines 18-19) and attached to oligonucleotides that can include capture probes (p 160, lines 29-31 through p161 line 1). Thus, Frenz teaches the use of a capture probe as encompassed by the claimed hydrogel-binding probe of claim 1, step (a).
Regarding claim 1 step (b), Frenz teaches the method further comprises a biological sample embedded in a hydrogel made up of hydrogel subunits (p 53, lines 10-15; see also Fig. 24). The biological sample may be immobilized (i.e. embedded) via cross-linking (p 53, lines 26-27). Frenz further teaches that acrydite moieties (i.e. hydrogel-binding moieties) can be modified to form chemical bonds capable of forming disulfide bonds (p160, lines 18-22), thus providing the crosslinking of capture probes to hydrogels via the hydrogel-binding moiety.
Regarding claim 1 step (c), Frenz teaches the method of reversing an acrydite moiety (i.e. hydrogel-binding moiety) bonds and releasing the attached species (i.e. analyte bound to the hydrogel-binding probe from step a ) (p 160, lines 22-24). Frenz further teaches an analyte can be transported to a capture probe affixed to a substrate ( p 238, lines 29-31). Frenz also teaches a substrate with a second affinity group (i.e. second binding moiety) which is directly bound to the substrate (p 182, lines 5-6). Frenz further teaches that that biotinylated oligonucleotides with sequence complementary to one or more analytes interest, or complements thereof (i.e. the hydrogel-binding probe from step a), can bind to the analyte(s) of interest and can be selected using biotinylation-strepavidin affinity (p 252, lines 29-31).
Regarding claim 1 step (d), Frenz teaches extending capture probes (e.g. hydrogel-binding probes) by generating cDNA from a captured (hybridized) RNA (p 276, lines 14-15) with the RNA acting as a template for extension (p 276, line 19). Frenz further teaches that a polynucleotide tail, e.g., a poly(A) tail (i.e. capture sequence), can be incorporated at the 3' end of the extended probe molecules (p 277, lines 20-21).
Regarding claim 1 step (e), Frenz teaches the use of two substrates, where the two substrates include (1) an array with capture probes on one substrate and (2) a biological sample on a different substrate (p235, lines 6-7). The substrate with the biological sample (2) may be permeabilized to allow analytes to interact with the capture probes on the other substrate (p236, lines 14-16). Frenz further teaches the use of a second substrate with an array of features (p 360, lines7-8 and see Fig. 22A), where features comprise a capture probe, wherein the capture probe comprises a spatial barcode and a capture domain (p 17, lines 15-16). Frenz teaches that the capture domain can comprise a poly(T) sequence that hybridizes to the capture sequence comprising a poly(A) nucleotides sequence (p 115, lines 26-28) as incorporated into the extended probe in claim 1 step (d).
Regarding claim 1 step (f), Frenz teaches extending the capture probe (hydrogel binding probe, here “extended probe”) (p 108, lines 17-22), using the capture domain of the capture probe as a primer (p109, lines 4-5) thus satisfying the requirement of step (f) for a capture probe to act as the template for generating a first strand.
Regarding claim 1 step (g), Frenz teaches determining (i) all or a part of the sequence of the analyte specifically bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample (p 14, lines 13-17).
Regarding claim 2, Frenz teaches synthesis of a second strand complementary to the extended capture probe (i.e. extended hydrogel binding probe) (p 295, lines 24-25
Regarding claim 3, Frenz teaches blocking the 3’ end of a capture probe not bound to the analyte (p21, lines 12-14). Capture probes of the array bind the extended hydrogel-binding probe and not the analyte, thus satisfying the requirements of claim 3.,
Regarding claim 4, Frenz teaches capture probes that include a cleavage domain, one or more functional domains and a unique molecular identifier in addition to a capture domain (p 79, lines 21-22)
Regarding claim 5, Frenz teaches the biological analyte comprises an mRNA (p 14, lines 1-2 and claim 90).
Regarding claim 6, Frenz teaches the use of multiple capture probes (p 101, lines 23-25) with embodiments that include a capture domain comprising a poly(T) sequence (p17, lines 17-18). Frenz further teaches that an analyte capture sequence comprises a poly(A) nucleic acid sequence (p 115, lines 26-27).
Regarding claim 7, Frenz teaches the use of an acrydite moiety that can bind to a bead (hydrogel) and capture probe (p 160, lines 13-19). Frenz further teaches that the capture probe can be attached to the bead (i.e. through the acrydite moiety) such that the capture probes retain a free 3’ end (p161, lines 3-5). Thus the acrydite moiety linking the capture probe to the bead must be at the 5’ end of the capture probe, satisfying the requirements of claim 7.
Regarding claim 8, Frenz teaches clearing the biological sample by permeabilizing (p 26, lines 10-13) after embedding and crosslinking (i.e. claim 1 step(b) and before releasing the analyte (i.e. claim 1 step (c) (see Fig. 24). Frenz also reports suitable agents for permeabilization (i.e. clearing) include proteases such as proteinase K (p 64, lines 7-11).
Regarding claim 10, Frenz teaches using RNase H to release RNA-templated ligation products (i.e. the RNA analyte bound to the extended probe) (p 255, lines 15-16).
Regarding claim 11, Frenz teaches treating the biological sample with a permeabilization reagent prior to contacting the substrate with the biological sample (p 9, lines 23-25 and claim 32).
Regarding claim 12, Frenz teaches that reversible cross-linking can allow dissociation of cross-linked polymers and for reversible attachment of a material (i.e. analyte) bound to the surface of a bead (i.e. the hydrogel) (p 159, lines 7-9).
Regarding claim 13, Frenz teaches analytes can be migrated from a sample to a substrate and that migration can be active (e.g. electrophoretic) (p 236, lines 29-30).
Regarding claim 14, Frenz teaches a hydrogel can act as diffusion resistant medium that can limit diffusivity of poly(A) transcripts (p 238, lines 12-14). Frenz further teaches that the biological sample may be immobilized (i.e. resisting migration) via cross-linking to the hydrogel (p 53, lines 26-27).
Regarding claim 15, Frenz teaches using reversible cross-linking to dissociate the cross-linked polymers of a hydrogel (p 159, lines 7-9), thus removing the agent decreasing migration of the analyte as required in claim 15. Frenz further teaches analyte migration can be active migration, using an electrophoretic transfer system, (p 80, lines 1-2).
Regarding claim 16, Frenz teaches a capture domain can be coupled to a magnetic particle (p312, line 17) and that analytes can be actively directed to the capture probes attached to a substrate using a magnetic field (p 29, lines 27-29).
Regarding claim 17, Frenz teaches negatively charged mRNA target analytes migrate towards a positively charged anode (p 243, lines5-6 and Fig. 15). Frenz further teaches an electric current can promote the directional migration of charged analytes towards capture probes on a substrate using electrophoresis (p240, lines 9-16).
Regarding claim 18, Frenz teaches creating a 3’ overhang to a capture probe (i.e. adding sequence to the 3’ end) using terminal deoxynucleotidyl transferase or poly(A) polymerase (p 146, lines 15-18).
Regarding claim 19, Frenz teaches extending capture probes adding nucleotides to the 3’ end using DNA polymerase or reverse transcriptase (p108, lines 18-31 and p 109 lines 1-3).
Regarding claim 20, Frenz teaches embodiments where the first affinity group or the second affinity group is biotin, and the other of the first affinity group or the second affinity group is streptavidin (p 183, lines 11-12). Frenz teaches hydrogel subunits include acrylamide and polyacrylamide (p227, line 20).
Regarding claim 21, Frenz teaches staining and imaging the biological sample before embedding the sample in a hydrogel (p 79, lines 4-10 and Fig. 3).
Response to Arguments against Claim Rejection - 35 U.S. C § 102
The response asserts that that the hydrogel-binding probes recited in claim 1(a) are distinct from capture probes recited in claim 1(e), and the capture probes of Frenz cannot be analogized as the hydrogel-binding probes of claim 1(a) (p. 9).
Applicant's arguments have been fully considered but are not persuasive.
It is acknowledged that the hydrogel-binding probes of instant claim 1(a) are distinct from the capture probes recited in claim 1(e). However, Frenz teaches a plurality of capture probes (p. 5, line 21) that encompass a broad genus of capture probes (Figs. 6-8, 10), encompassing both the hydrogel-binding probe of claim 1(a) and the capture probe of claim 1(e).
There is no limitation in the claim that excludes the claimed hydrogel-binding probes and capture probe from being species of the capture probes of Frenz. Different species of capture probes may be used at different steps of the method. The instant claim does not limit the hydrogel-binding or capture probes to a specific sequence or structure.
Frenz teaches capture probes can be attached to a surface or unattached (p. 29, lines 16-17); encapsulated within, embedded within, or layered on a surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel) (p. 29, lines 20-24). The capture probes of Frenz comprise: spatial barcodes, capture domains, and functional sequences, and varied combinations of these elements, including extension by binding to an analyte capture agent (Figs. 8-10).
Thus, a capture probe performing the function of hydrogel binding reads on the hydrogel-binding probes of claim 1(a), while a capture probe attached to an array that functions to capture an extended probe reads on the capture probes of claim 1(e).
The response asserts that the biotinylated oligonucleotides described in Frenz are included in a section entitled "Enrichment of Captured Analytes after Capture." Thus, read in context, the analytes in Frenz are captured by capture probes, optionally amplified, and then subsequently contacted with a different oligonucleotide that includes a biotin moiety (Specification, page 249, line 4 to page 251, line 1), i.e. Frenz does not describe capture probes with biotin. (p. 9).
Applicant's arguments have been fully considered but are not persuasive.
As written, the claim only recites the limitation “wherein a hydrogel-binding probe – comprises -- (ii) a first binding moiety” but does not require when the binding moiety is added. The claim does not require when or how a binding moiety is made part of the hydrogel-binding probe. Further, the claim does not restrict in any way how the elements (i), (ii), and (iii) are arranged or configured or the timing of inclusion in the hydrogel-binding probe, simply that the elements, including the first binding moiety, must be there at some point. It is noted that the binding moiety is not required in claim 1(a)
Frenz also states th.at the specific embodiments described in their specification are intended to illustrate and not limit the scope of the invention (p. 395, lines 11-13).
The response asserts that the acrydite moiety of Frenz is present in a bead and not a hydrogel binding probe comprising (i) a first capture domain, wherein the first capture domain binds to the analyte; (ii) a first binding moiety; and (iii) a hydrogel-binding moiety as required by the present claims (p. 9-10).
Applicant's arguments have been fully considered but are not persuasive.
As written, the claim only recites the limitation “wherein a hydrogel-binding probe – comprises -- (iii) a hydrogel-binding moiety” but does not require when a hydrogel-binding moiety is added. The claim does not require when or how a hydrogel-binding moiety is made part of the hydrogel-binding probe. Further, the claim does not restrict in any way how the elements (i), (ii), and (iii) are arranged or configured or the timing of inclusion in the hydrogel-binding probe, simply that the elements, including the hydrogel-binding moiety, must be there at some point.
There is no limitation in the claim that excludes the claimed probe (comprising a hydrogel-binding moiety) from being attached to a bead.
Further, although Frenz refers to acrydite moieties attached to beads, acrydite moieties are not limited to beads. As described above in the 102 rejection, the acrydite moieties (i.e. a hydrogel-binding moiety) can be attached to capture probes: “The acrydite moieties can be attached to a nucleic acid molecule (e.g., an oligonucleotide) which can include a priming sequence (e.g., a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more capture probes” (p. 160 lines 29-31 to p. 161, line 1).
Thus the acrydite (hydrogel-binding moiety) attached to a capture probe has structural features encompassed by the broad scope of the claimed hydrogel-binding probe (comprising a hydrogel-binding moiety and reads on the limitation in claim 1(a) “wherein a hydrogel-binding probe – comprises -- (iii) a hydrogel-binding moiety”.
The response asserts that, with respect to claim 1(e), Frenz does not teach or suggest a method where a substrate comprising the extended probe generated in (d) and attached to a substrate via interaction between the first and second binding moieties binds the second capture domain of the capture probe on the array as presently required by the claims (p. 10). The response supports the argument with Figure 3(e) of the instant application, a non-limiting embodiment where the extended probe binds to the second capture domain of the capture probe of the array (p. 11).
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., Figure 3(e)) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
As described above, Frenz teaches the use of two substrates (an array of capture probes and a substrate comprising the biological sample, thus comprising the extended probe of the biological sample). As cited in the 102 rejection, the capture probes of the array capture the poly(A) tail of the extended probe, which read on the requirements of claim 1(e).
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.
Claim 9 remain/is rejected under 35 U.S.C. 103 as being unpatentable over Frenz et al. (WO2020123316A2, on IDS dated 04/21/2023) in view of Hytönen et al. (Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins. 2005. Journal of Biological Chemistry. 280(11): 10228-10233. On IDS dated 4/21/2023).
The teachings of Frenz as they relate to claim 1 are stated above under 102 rejection in this office action.
Regarding claim 9, Frenz does not teach the plurality of second binding moieties and the first binding moiety of the hydrogel-binding probe are resistant to degradation by proteinase K.
Hytönen teaches an engineered form avidin (second binding moieties) that binds to biotin (a first binding moiety) (p 10228, col 1 abstract). Hytönen further teaches the engineered avidin is protected from digestion by proteinase K (p 10230, col. 1). Hytönen states that the engineered avidin produces a more stable protein useful in applications utilizing extreme conditions in applications such as PCR, (p 10233, col. 1).
It would have been obvious to one with ordinary skill in the art before the effective filing date of the claimed invention to use the engineered binding moieties of Hytönen with the method of Frenz to arrive at the instantly claimed method with a reasonable expectation of success. The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method that protected the hydrogel-binding probes and capture probes of Frenz from being dissociated by the permeabilization step of Frenz, thereby allowing targeted permeabilization of only non-protected elements and leaving the protected probes bound to analytes. In addition, it would have been obvious to the ordinary artisan that the known techniques of the cited prior art could have been combined with predictable results because it depends only on substituting a different version of binding moieties.
Response to Arguments against Claim Rejection - 35 U.S. C § 103
The response asserts that the cited art does not disclose a method comprising at least (a) and (e) of claim 1 including: (a) contacting the biological sample with a plurality of hydrogel-binding probes, wherein a hydrogel-binding probe of the plurality of hydrogel-binding probes comprises (i) a first capture domain, wherein the first capture domain binds to the analyte; (ii) a first binding moiety; and (iii) a hydrogel-binding moiety; and ( e) contacting the substrate comprising the extended probe with an array comprising a plurality of capture probes, wherein a capture probe comprises (i) a second capture domain, wherein the second capture domain binds the capture sequence of the extended probe, and (ii) a spatial barcode (p. 11).
The response further asserts that Hytonen fails to cure the deficiencies of Frenz and that the cited art (considered alone or together) does not teach or suggest all the limitations of the claims and there is no motivation for one of ordinary skill in the art to modify the cited art to arrive at the present claims with any reasonable expectation of success (p. 12).
Applicant's arguments have been fully considered but are not persuasive.
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).
It is the Examiner's position that Hytönen does teach the limitations of claim 9 and motivations to modify the cited art to arrive at the present claims as outlined in the rejection of claim 9 provided above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
(I). (i). Claims 1, 4-6, and 10-11 are rejected on the ground of nonstatutory double patenting as being unpatentable over, in the alternative, claims 1, 17,18, 21, and 23-24 of U.S. Patent No. 11,560,593, over claims 1, 15, 19, 21-22, and 27 of U.S. Patent No. 11,680,260, over claims 1, 10, and 12 of U.S. Patent No. 11,702,698, over claims 1, 16, and 17 of U.S. Patent No. 11,732,300, over claims 1,2, and 19-20 of U.S. Patent No. 12,286,673, over claims 1,2, and 19-20 of U.S. Patent No. 11,753,673, over claims 1 and 6 of U.S. Patent No. 11,840,724, over claims 1, 6, 7, 9-11,13,15, 17, and 19-21of U.S. Patent No. 11,753,675, over claims 1, 7-8, and 10-12 of U.S. Patent No. 12,031,177, over claims 1, 9, 19-20, and 25-26 of U.S. Patent No. 12,071,655. Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims substantially anticipate the identified claims of this application.
The claims of the instant application and the patents cited above are all drawn to methods for determining the location of an analyte in a sample, the methods requiring steps which encompass the claims of the instant application.
Regarding instant claim 1, claim 1 of the ‘593 patent requires a method comprising: (a) providing an array comprising a plurality of capture probes affixed to the array, wherein a capture probe comprises: (i) a spatial barcode and (ii) a capture domain; (b) contacting a plurality of first probes and second probes to a biological sample on the array, wherein the first probes and second probes target a plurality of nucleic acids in the biological sample, wherein a first probe comprises a first sequence that is substantially complementary to a first target sequence of the target nucleic acid; wherein a second probe comprises: a second sequence that is substantially complementary to a second target sequence of the target nucleic acid; a third sequence that is substantially complementary to a third target sequence of the target nucleic acid; and wherein the first probe or the second probe comprises a capture probe capture domain that is complementary to all or a portion of the capture domain of the capture probes affixed to the array; (c) hybridizing the first probe to the first sequence and the second probe to the second sequence and the third sequence; (d) generating a ligation product by ligating the first probe and the second probe; (e) hybridizing the ligation product to the capture domain of the capture probe; and (f) determining (i) all or part of the sequence of the ligation product hybridized to the capture domain, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the target nucleic acid in the biological sample.
Regarding instant claim 1, claim 1 of the ‘260 patent requires a method comprising: (a) providing the biological sample, wherein the biological sample is a tissue sample that was previously frozen and then fixed in a fixative selected from acetone, methanol, or an acetone-methanol mixture; (b) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences of the nucleic acid analyte, and wherein the second probe comprises a capture probe binding domain; (c) coupling the first probe and the second probe, thereby generating a connected probe; (d) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (e) releasing the connected probe from the nucleic acid analyte when at least a portion of the biological sample is aligned with at least a portion of the array; and (f) hybridizing the connected probe to the capture domain of the capture probe. Claim 19 of the ‘260 patent further requires determining (i) all or a part of the sequence of the connected probe, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine a location of the nucleic acid analyte in the biological sample.
Regarding instant claim 1, claim 1 of the ‘698 patent requires a method comprising: (a) contacting the biological sample with a first substrate; (b) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein the second probe comprises a capture binding domain that hybridizes to a capture domain of a capture probe, wherein the capture probe is affixed to the first substrate or wherein the capture probe is affixed to a second substrate, wherein the capture probe further comprises a spatial barcode, and wherein a portion of the capture binding domain is hybridized to a blocking probe; (c) ligating the first probe and the second probe, thereby creating a ligation product, wherein the ligation product comprises a sequence that is substantially complementary to the nucleic acid analyte; (d) releasing: (i) the ligation product from the nucleic acid analyte, and (ii) the blocking probe from the capture binding domain; (e) hybridizing the capture binding domain to the capture domain; and (f) determining (i) all or part of the sequence of the ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify a location of the nucleic acid analyte in the biological sample. Claim 12 of the ‘698 patent further requires wherein the capture probe is on the second substrate, wherein the second substrate comprises a plurality of capture probes, and wherein each capture probe of the plurality of capture probes comprises the capture domain.
Regarding instant claim 1, claim 1 of the ‘300 patent requires a method comprising (a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode; (b) hybridizing the analyte to the capture domain, thereby generating a capture analyte; and (c) contacting the captured analyte to a bridging oligonucleotide, wherein the bridging oligonucleotide comprises: (i) a capture-probe-binding sequence, and (ii) an analyte-binding sequence; (d) extending the bridging oligonucleotide using the analyte as a template to generate an extended capture probe comprising (i) the analyte, or a complement thereof, and (ii) the spatial barcode or a complement thereof; and (e) determining (i) all or a part of the sequence of the analyte, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the location of the analyte in the biological sample.
Regarding instant claim 1, claim 1 of the ‘6673 patent requires a method comprising: (a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode; (b) hybridizing an analyte to the capture domain, thereby generating a capture analyte; and (c) contacting the captured analyte to a bridging oligonucleotide, wherein the bridging oligonucleotide comprises: (i) a capture-probe-binding sequence, and (ii) an analyte-binding sequence; and (d) extending the bridging oligonucleotide using the analyte as a template to generate an extended capture probe comprising (i) the analyte, or a complement thereof, and (ii) the spatial barcode or a complement thereof. Claim 2 of the ‘6673 patent further requires sequencing (i) all or a part of the sequence of the analyte, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the sequences of (i) and (ii) to determine location of the analyte in the biological sample.
Regarding instant claim 1, claim 1 of the ‘3673 patent requires a method comprising: (a) disposing the biological sample onto an array at a first area, wherein the array comprises a plurality of capture probes, wherein: a first capture probe of the plurality of capture probes comprises a first spatial barcode and a capture domain, wherein the first capture probe is comprised in the first area of the array; and a second area of the array comprises a second capture probe of the plurality of capture probes, wherein the second capture probe comprises a second spatial barcode and the capture domain, wherein the second area is not covered by the biological sample disposed on the array; (b) contacting the second area of the array with a solution comprising terminal deoxynucleotidyl transferase and one or more dideoxynucleotides, such that a dideoxynucleotide is incorporated into the capture domain of the second capture probe; (c) permeabilizing the biological sample, such that the capture domain of the first capture probe hybridizes to the target nucleic acid from the biological sample; and (d) determining (i) a sequence of the first spatial barcode of the first capture probe, or a complement thereof, and (ii) all or a portion of a sequence of the target nucleic acid, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the target nucleic acid in the biological sample. Claim 4 of the ‘673 patent further requires extending a 3′ end of the first capture probe of the first area of the array using the target nucleic acid as a template, thereby generating an extended capture probe.
Regarding instant claim 1, claim 1 of the ‘724 patent requires a method comprising: (a) disposing the biological sample onto an array at a first area, wherein the array comprises a plurality of capture probes, wherein: a first capture probe of the plurality of capture probes comprises a first spatial barcode and a capture domain, wherein the first capture probe is comprised in the first area of the array; and a second area of the array comprises a second capture probe of the plurality of capture probes, wherein the second capture probe comprises a second spatial barcode and the capture domain, wherein the second area is not covered by the biological sample disposed on the array; (b) contacting the second area of the array with a solution comprising terminal deoxynucleotidyl transferase and one or more dideoxynucleotides, such that a dideoxynucleotide is incorporated into the capture domain of the second capture probe; (c) contacting the biological sample with an analyte capture agent, wherein the analyte capture agent comprises an analyte binding moiety that specifically binds the protein and an oligonucleotide comprising an analyte capture sequence and an analyte-binding moiety barcode; (d) hybridizing the capture domain of the first capture probe to the oligonucleotide of the analyte capture agent; and (e) determining (i) a sequence of the first spatial barcode of the first capture probe, or a complement thereof, and (ii) a sequence of the analyte-binding moiety barcode, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the protein in the biological sample.
Regarding instant claim 1, claim 1 of the ‘675 patent requires a method comprising: (a) contacting the biological sample with a first substrate; (b) adding a hydrogel matrix to the biological sample on the first substrate, thereby isometrically expanding the biological sample; (c) hybridizing the analyte to a capture probe comprising a capture domain and a spatial barcode, wherein the capture probe is affixed to an array; (d) extending the capture probe using the analyte as a template; and (e) determining (i) the spatial barcode or a complement thereof, and (ii) all or a portion of the analyte from the biological sample or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample. Claims 6 and 7 further require capture probe is affixed to the array on the first substrate (claim 6) and capture probe is affixed to the array on the first substrate (claim 7). Claim 13 further requires extending the capture probe using the analyte as a template, thereby generating an extended capture probe. Claim 15 further requires the determining step comprises sequencing.
Regarding instant claim 1, claim 1 of the ‘177 patent requires a method comprising: (a) providing a first substrate comprising a plurality of first capture probes, wherein a first capture probe of the plurality of first capture probes comprises a first capture domain, the first substrate comprising the biological sample mounted thereon; (b) providing a second substrate on the opposite side of the first substrate relative to the biological sample, thereby sandwiching the first substrate, the biological sample, and the second substrate, wherein the second substrate comprises a plurality of second capture probes, wherein a second capture probe of the plurality of second capture probes comprises (i) a spatial barcode and (ii) a second capture domain; (c) hybridizing the analyte to the second capture domain and hybridizing a second analyte to the first capture domain; and (d) determining sequences of (i) the spatial barcode, or a complement thereof, and (ii) all or a portion of the analyte captured on the second capture domain, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the analyte in the biological sample.
Regarding instant claim 1, claim 1 of the ‘655 patent requires a method comprising: (a) providing a first substrate and the biological sample mounted thereon, wherein the first substrate comprises a plurality of first capture probes, wherein a first capture probe of the plurality of first capture probes comprises (i) a first spatial barcode and (ii) a first capture domain; (b) aligning a second substrate on the opposite side of the first substrate relative to the biological sample, thereby sandwiching the biological sample between the first and the second substrate, wherein the second substrate comprises a plurality of second capture probes, wherein a second capture probe of the plurality of second capture probes comprises (i) a second spatial barcode and (ii) a second capture domain; (c) adding a reagent medium comprising a permeabilization buffer to the biological sample prior to aligning, thereby promoting migration of a first intermediate agent and a second analyte or a second intermediate agent from the biological sample to the first substrate and/or the second substrate, wherein the first intermediate agent is generated by hybridizing a first probe and a second probe to a first analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to adjacent sequences of the first analyte, and wherein the second probe comprises a first capture probe binding domain; and coupling the first probe and the second probe, thereby generating a first connected probe; (d) hybridizing the first intermediate agent to the first capture domain, and hybridizing the second analyte or the second intermediate agent to the second capture domain; and (e) (A) determining (i) the sequence of the first spatial barcode, or a complement thereof, and (ii) all or a portion of the sequence of the first intermediate agent, or a complement thereof, and using the sequences of (i) and (ii) to determine the location of the first analyte in the biological sample, and (B) determining (iii) the sequence of the second spatial barcode, or a complement thereof, and (iv) all or a portion of the sequence of the second analyte or the second intermediate agent, or a complement thereof, and using the sequences of (iii) and (iv) to determine the location of the second analyte in the biological sample. Claim 9 of the ‘655 patent further requires generating a first extended capture probe using the first analyte or first intermediate agent as a template.
Regarding instant claim 4, claim 21 of the ‘260 patent requires the capture probes comprises one or more functional domains, a unique molecular identifier (UMI), a cleavage domain, or combinations thereof.
Regarding instant claim 4, claim 19 of the ‘675 patent requires the capture probes further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and combinations thereof.
Regarding instant claim 4, claim 10 of the ‘177 patent requires the second capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, or a combination thereof.
Regarding instant claim 5, claims 23 and 24 of the ‘593 patent require the target nucleic acid comprises RNA, wherein the RNA is an mRNA.
Regarding instant claim 5, claim 22 of the ‘260 patent requires the nucleic acid analyte is RNA.
Regarding instant claim 5, claim 10 of the ‘698 patent requires the nucleic acid analyte is RNA.
Regarding instant claim 5, claim 16 of the ‘300 patent requires the analyte is mRNA.
Regarding instant claim 5, claim 19 of the ‘6673 patent requires the analyte is mRNA.
Regarding instant claim 5, claims 20 and 21 of the ‘675 patent require the analyte comprises DNA or RNA (claim 20) and the RNA is mRNA (claim 21).
Regarding instant claim 5, claims 11 and 12 of the ‘177 patent require the analyte and the second analyte are RNA molecules (claim 11) and the RNA molecules are mRNA molecules (claim 12).
Regarding instant claim 5, claims 19 and 20 of the ‘655 patent require the analyte and the second analyte are RNA molecules (claim 19) and the RNA molecules are mRNA molecules (claim 20).
Regarding instant claim 6, claim 17 of the ‘300 patent requires the capture domain comprises a poly(T) sequence.
Regarding instant claim 6, claim 20 of the ‘6673 patent requires the capture domain comprises a poly(T) sequence.
Regarding instant claim 6, claim 17 of the ‘675 patent requires the capture domain comprises a poly(T) sequence.
Regarding instant claim 6, claims 7 and 8 of the ‘177 patent requires the first capture domain comprises a poly-thymine (poly(T)) sequence (claim 7) and the second capture domain comprises a poly-thymine (poly(T)) sequence (claim 8).
Regarding instant claim 6, claim 26 of the ‘655 patent requires the first capture domain and/or the second capture domain comprises a poly(T) sequence.
Regarding instant claim 10, claims 17 and 18 of the ‘593 patent require releasing the ligation product from the target nucleic acid, wherein releasing comprises contacting the biological sample with an endoribonuclease, wherein the endoribonuclease is an RNase H enzyme.
Regarding instant claim 10, claim 15 of the ‘260 patent requires the agent for releasing the connected probe comprises an RNase.
Regarding instant claim 11, claim 21 of the ‘593 patent requires the biological sample was previously stained.
Regarding instant claim 11, claim 27 of the ‘260 patent requires further comprising imaging and/or destaining the biological sample.
Regarding instant claim 11, claim 7 of the ‘3673 patent requires further comprising staining, and/or imaging the biological sample.
Regarding instant claim 11, claim 6 of the ‘724 patent requires further comprising staining, and/or imaging the biological sample.
Regarding instant claim 11, claims 9,10 and 11 of the ‘675 patent require further comprising imaging the biological sample (claim 9), staining the biological sample (claim 10), and permeabilizing the biological sample (claim 11).
Regarding instant claim 11, claim 25 of the ‘655 patent requires further comprising staining, imaging, or destaining the biological sample, or a combination thereof.
(ii). Claims 2-3, 7-8, and 12-21 are rejected on the ground of nonstatutory double patenting as being unpatentable over, in the alternative, claims 1, 17,18, 21, and 23-24 of U.S. Patent No. 11,560,593, over claims 1, 15, 19, 21-22, and 27 of U.S. Patent No. 11,680,260, over claims 1, 10, and 12 of U.S. Patent No. 11,702,698, over claims 1, 16, and 17 of U.S. Patent No. 11,732,300, over claims 1,2, and 19-20 of U.S. Patent No. 12,286,673, over claims 1,2, and 19-20 of U.S. Patent No. 11,753,673, over claims 1 and 6 of U.S. Patent No. 11,840,724, over claims 1, 6, 7, 9-11,13,15, 17, and 19-21of U.S. Patent No. 11,753,675, over claims 1, 7-8, and 10-12 of U.S. Patent No. 12,031,177, over claims 1, 9, 19-20, and 25-26 of U.S. Patent No. 12,071,655 in view of Frenz et al. (WO2020123316A2, on IDS dated 04/21/2023. Filed 12/06/2019 and published 06/18/2020).
Regarding instant claims 2-3, 7-8, and 12-20, the claims of the ‘above patents do not require the limitations in the instant claims.
The teachings of Frenz as they relate to these claims are given previously in this office action and are fully incorporated here.
(iii). Claim 9 is rejected on the ground of nonstatutory double patenting as being unpatentable over, in the alternative, claims 1, 17,18, 21, and 23-24 of U.S. Patent No. 11,560,593, over claims 1, 15, 19, 21-22, and 27 of U.S. Patent No. 11,680,260, over claims 1, 10, and 12 of U.S. Patent No. 11,702,698, over claims 1, 16, and 17 of U.S. Patent No. 11,732,300, over claims 1,2, and 19-20 of U.S. Patent No. 12,286,673, over claims 1,2, and 19-20 of U.S. Patent No. 11,753,673, over claims 1 and 6 of U.S. Patent No. 11,840,724, over claims 1, 6, 7, 9-11,13,15, 17, and 19-21of U.S. Patent No. 11,753,675, over claims 1, 7-8, and 10-12 of U.S. Patent No. 12,031,177, over claims 1, 9, 19-20, and 25-26 of U.S. Patent No. 12,071,655 in view of Hytönen et al. (Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins. 2005. Journal of Biological Chemistry. 280(11): 10228-10233. On IDS dated 4/21/2023).
Regarding instant claim 9, the claims of the above patents do not require the plurality of second binding moieties and the first binding moiety of the hydrogel-binding probe are resistant to degradation by proteinase K.
The teachings of Hytönen as they relate to these claims are given previously in this office action and are fully incorporated here.
(II). (i). Claims 1, 4-6, and 11 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 213-214, 216, 224-225, 226-228, and 235-236 of copending Application No. 17/312,339 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims substantially anticipate the identified claims of this application.
The claims of the instant application and copending application are each drawn to methods for determining the location of an analyte in a sample, the methods teaching steps which encompass the claims of the instant application.
Regarding instant claim 1, copending claim 213 teach a method comprising: (a) providing an a substrate, wherein the substrate comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;(b) contacting the substrate with the biological sample, wherein the capture domain of the capture probe hybridizes to the analyte from the biological sample to generate a captured analyte;(c) contacting a plurality of fluorescently labeled probes with the captured analyte such that the plurality of fluorescently labeled probes hybridizes to the cap