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 .
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-2, 6-34) in the reply filed on 09/10/2025 is acknowledged.
Claim Objections
Claims 7-34 are objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only and/or cannot depend from any other multiple dependent claim(s). See MPEP § 608.01(n). Accordingly, the claims 7-34 have not been further treated on the merits.
Claim 6 is objected to because of the following informalities: claim 6 depends from canceled claims 3-5. Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 2, and 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Daugharthy et al. WO 2017/079406 A1 in view of Fonnum et al. US 2018/0215852 A1.
Regarding claim 1, Daugharthy et al. discloses a method of forming a three dimensional (3D) sequencing substrate (abstract .. Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.. pg 3 In 21-24 .. The nucleic acids may be co-polymerized
with the matrix material or cross-linked to the matrix material or both.. the 3D substrate is formed) comprising: (a) amplifying a plurality of nucleic acid molecules from a sample where the nucleic acid molecule from the plurality of nucleic acid molecules or an amplicon thereof is coupled to the substrate (pg 4 In 12-16 .. The nucleic acids can be amplified and sequenced, if desired, in situ thereby providing positional information of the nucleic acids within the cell or tissue.. pg 4 In 20-24 .. In this manner, a three-dimensional matrix of covalently bound nucleic acids of any desired sequence is provided. Each nucleic acid has its own three-dimensional coordinates within the matrix
material and each nucleic acid represents information. According to one aspect, individual nucleic acids, such as DNA or RNA can be amplified and sequenced in situ, i.e., within the matrix.. pg 27 In 8-11 .. In certain embodiments, nucleic acids are those found naturally in a biological sample, such as a cell or tissue..) (b) forming a three dimensional (4U) sequencing substrate (abstract .. Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.. pg 3 In 21-24 .. The nucleic acids may be co-polymerized
with the matrix material or cross-linked to the matrix material or both.. the 3D substrate is formed).
Daugharthy et al. does not specifically disclose wherein the nucleic acids are in a plurality of partitions, wherein a partition of the plurality of partitions comprises a nucleic acid molecule from the plurality of nucleic acid molecules and a substrate, and wherein amplification couples the nucleic acid molecule from the plurality of nucleic acid molecules or an amplicon thereof to the substrate; and where substrate forming is from the plurality of partitions.
Daugharthy et al. discloses polymerizing monomers to form the 3D substrate wherein the nucleic acids are attached prior to polymerization such that they are copolymerized (pg 3 In 19-24 .. Such methods include making a three-dimensional matrix including nucleic acids covalently bound into a matrix or into or to a matrix material. The nucleic acids may be co-polymerized with the matrix material or cross-linked to the matrix material or both..).
Fonnum et al. discloses a method of forming a sequencing substrate (para [0010] .. The invention is based in part on an appreciation that seed particles comprising hydrophilic oligomers may be used in a novel process for forming cross - linked monodisperse polymeric particles.. para [0018] .. An eighth aspect comprises the use of monodisperse crosslinked hydrogel polymer particles in nucleic acid amplification and / or oligonucleotide sequencing..) wherein the substrate is formed by starting with a plurality of partitions (para [0015] .. A fifth aspect provides a method of forming monodisperse crosslinked hydrogel polymer particles. The method comprises forming a solution (4 ) of at least 2 % wt of a hydrophilic vinylic monomer in an aqueous solution , the aqueous solution also comprising a crosslinker comprising at least two vinyl groups ; forming a solution ( b ) of stabilizer in an organic solvent , wherein the organic solvent is
not miscible in water , and wherein at least one of solution ( a ) and solution ( b ) comprises a radical initiator ; mixing solutions ( a ) and (b ) to form a water - in - oil emulsion ( c ) and adding monodisperse seed particles to the emulsion ; allowing the monodisperse seed particles to form swollen particles in the emulsion ; and polymerizing the swollen particles to form the monodisperse crosslinked hydrogel
polymer particles.. see instant specification para [0042] ..In an example for nucleic acid analysis of a sample, a plurality of single molecules (e.g., nucleic acid molecules of a sample) are physically separated into individual compartments or partitions. Such partitions can be droplets such as emulsion droplets.. emulsions comprise partitions) and with a plurality of nucleic acids wherein amplification couples the nucleic acid molecule from the plurality of nucleic acid molecules or an amplicon thereof to the substrate (para [0192] .. In embodiments, polymeric particles from an emulsion-breaking procedure may be collected and washed in preparation for sequencing.
Collection may be conducted by contacting biotin moieties (e.g., linked to amplified polynucleotide templates which are attached to the polymeric particles) with avidin moieties, and separation away from polymeric particles lacking biotinylated templates. Collected polymer particles that carry double-stranded template polynucleotides may be denatured to yield single-stranded template polynucleotides for sequencing.. para [0193] .. When a nucleotide is captured by a polymerase and added to the end of an extending primer, a label of the nucleotide may fluoresce, indicating which type of nucleotide is added. These and other sequencing methods described herein may be
combined with methods for nucleic acid amplification. For example, in the methods for nucleic acid amplification described herein with steps (a)-(g), the methods may comprise a step (h) of sequencing by detecting the nucleotide addition.. a DNA primer is attached to the particle and a target nucleic acid hybridizes and is amplified, coupling it to the particle). Fonnum et al. does not specifically disclose wherein the amplification occurs in the partitions. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to attach the nucleic acids using an amplification technique prior to the final formation of the substrate in order to have finer control over the location. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the method of 3D sequencing in a polymer substrate disclosed by Daugharthy et al. with the polymer particles used for sequencing which are formed via partition polymerization disclosed by Fonnum et al. in order to tune the properties of the substrate.
Regarding claim 2, Daugharthy et al. discloses a method of forming a three dimensional sequencing substrate (abstract .. Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.. pg 3 In 21-24 .. The nucleic acids may be co-polymerized with the
matrix material or cross-linked to the matrix material or both.. the 3D substrate is formed) comprising: (a) distributing a plurality of nucleic acid molecules of a substrate of a plurality of substrates (pg 4 In 12-16 .. The nucleic acids can be amplified and sequenced, if desired, in situ thereby providing positional information of the nucleic acids within the cell or tissue.. pg 4 In 20-24 .. In this manner, a three-dimensional matrix of covalently bound nucleic acids of any desired sequence is provided. Each nucleic acid has its own three-dimensional coordinates within the matrix material and each nucleic acid represents information. According to one aspect, individual nucleic acids, such as DNA or RNA can be amplified and sequenced in situ, i.e., within the matrix..); (b) coupling the nucleic acid molecule of the plurality of nucleic acid molecules to the substrate of the plurality of substrates to form a substrate conjugate of a plurality of substrate conjugates, thereby generating the plurality of substrate conjugates (pg 4 In 12-16 .. The nucleic acids can be amplified and sequenced, if desired, in situ thereby providing positional information of the nucleic acids within the cell or tissue.. pg 4 In 20-24 .. In this manner, a three-dimensional matrix of covalently bound nucleic acids of any desired sequence is provided. Each nucleic acid has its own three dimensional
coordinates within the matrix material and each nucleic acid represents information. According to one aspect, individual nucleic acids, such as DNA or RNA can be amplified and sequenced in situ, i.e., within the matrix..); and (c) forming a three dimensional sequencing substrate (abstract .. Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.. pg 3 In 21-24 .. The nucleic acids may be co-polymerized with the matrix material or cross-linked to the matrix material or both.. the 3D substrate is formed).
Daugharthy et al. does not disclose the nucleic acids in the sample are put into a plurality of partitions, wherein a partition of the plurality of partitions comprises a nucleic acid molecule of the plurality of nucleic acid molecules and or wherein the coupling occurs in the partition of the plurality of partitions such that the conjugates are in the plurality of partitions and the forming thus occurs from the plurality of partitions.
Daugharthy et al. discloses polymerizing monomers to form the 3D substrate wherein the nucleic acids are attached prior to polymerization such that they are copolymerized (pg 3 In 19-24 .. Such methods include making a three-dimensional matrix including nucleic acids covalently bound into a matrix or into or to a matrix material. The nucleic acids may be co-polymerized with the matrix material or cross-linked to the matrix material or both..).
Fonnum et al. discloses a method of forming a sequencing substrate (para [0010] .. The invention is based in part on an appreciation that seed particles comprising hydrophilic oligomers may be used in a novel process for forming cross - linked monodisperse . polymeric particles.. para [0018] .. An eighth aspect comprises the use of monodisperse crosslinked hydrogel! polymer particles in nucleic acid amplification and / or oligonucleotide sequencing..) wherein the substrate is formed by starting with a plurality of partitions (para [0015] .. A fifth aspect provides a method of forming monodisperse crosslinked hydrogel polymer particles . The method comprises forming a solution ( a ) of at least 2 % wt of a hydrophilic vinylic monomer in an aqueous solution , the aqueous solution also comprising a crosslinker comprising at least two vinyl groups ; forming a solution (b ) of stabilizer in an organic solvent , wherein the organic solvent is not miscible in water , and wherein at least one of solution (a ) and solution ( b ) comprises a radical initiator ; mixing solutions ( a ) and (b ) to form a water - in - oil emulsion (c ) and adding monodisperse seed particles to the emulsion ; allowing the monodisperse seed particles to form swollen particles in the emulsion ; and polymerizing the swollen particles to form the monodisperse crosslinked hydrogel polymer particles.. see instant specification para [0042] ..In an example for nucleic acid analysis of a sample, a plurality of single molecules (e.g., nucleic acid molecules of a sample) are physically separated into individual compartments or partitions. Such partitions can be droplets such as emulsion droplets.. emulsions comprise partitions) and with a plurality of nucleic acids wherein amplification couples the nucleic acid molecule from the plurality of nucleic acid molecules or an amplicon thereof to the substrate (para [0192] .. In embodiments, polymeric particles from an emulsion-breaking procedure may be collected and washed in preparation for sequencing. Collection may be conducted by contacting biotin moieties (e.g., linked to amplified polynucleotide templates which are attached to the polymeric particles) with avidin moieties, and separation away from polymeric particles lacking biotinylated templates. Collected polymer particles that carry double-stranded template polynucleotides may be denatured to yield single-stranded template polynucleotides for sequencing... para [0193] .. When a nucleotide is captured by a polymerase and added to the end of an extending primer, a label of the nucleotide may fluoresce, indicating which type of nucleotide is added. These and other sequencing methods described herein may be combined with methods for nucleic acid amplification. For example, in the methods for nucleic acid amplification described herein with steps (a)-(g), the methods may comprise a step (h) of sequencing by detecting the nucleotide addition.. a DNA primer is attached to the particle and a target nucleic acid hybridizes and is amplified, coupling it to the particle).
Fonnum et al. does not specifically disclose wherein the amplification occurs in the partitions. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to attach the nucleic acids using an amplification technique prior to the final formation of the substrate in order to have finer control over the location. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the method of 3D sequencing in a polymer substrate disclosed by Daugharthy et al. with the polymer particles used for sequencing which are formed via partition polymerization disclosed by Fonnum et al. in order to tune the properties of the substrate.
Regarding claim 6, Daugharthy et al. in view of Fonnum et al. discloses claim 2. Daugharthy et al. in view of Fonnum et al. does not disclose the method of claim 2, further comprising, prior to (a), (b), or (c), amplifying the plurality of nucleic acid molecules in the plurality of partitions. Daugharthy et al. discloses polymerizing monomers to form the 3D substrate wherein the nucleic acids are attached prior to polymerization such that they are copolymerized (pg 3 In 19-24 .. Such methods include making a three-dimensional matrix including nucleic acids covalently bound into a matrix or into or to a matrix material. The nucleic acids may be co-polymerized with the matrix material or cross-linked to the matrix material or both..). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to attach the nucleic acids using an amplification technique prior to the final formation of the substrate in order to have finer control over the location.
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/JEZIA RILEY/ Primary Examiner, Art Unit 1681 24 November 2025