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 .
Office Action: Notice
This Office Action is in response to the Pre-Appeal Brief Review, in which a decision was made to reopen prosecution (5/28/2026). Any objection or rejection of record in the previous Office Action, mailed 2/27/2026, which is not addressed in this action has been withdrawn in light of Applicants' amendments and/or arguments. This is a non-final office action.
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on 7/19/2023 is acknowledged. Claims 24-30 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Group 2, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7/19/2023. Thus, claims 1-4, 9, 11-17 and 21-23 and 31 are under examination (4/20/2026).
Claim Status
Applicant amended claims 1-2 (4/20/2026). Claims 5-8, 10, and 18-20 were previously cancelled (4/20/2026). No new matter was added. Thus, claims 1-4, 9, 11-17 and 21-23 and 31 are under examination.
Priority
Claims 1-4, 9, 11-17 and 21-23 receive a priority date of 9/15/2020, the filing date of US Provisional No. 63078754.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
The Information Disclosure Statement from 2/26/2026 is also being considered.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms “Triton-X” (p. 3, 31), “SplintR ligase” (p. 14), “Ion Torrent” (p. 17), “PGM” (p. 17), “Illumina” (p. 17), “PacBio” (p. 17), “Oxford Nanopore” (p. 17), “Kimwipe” (p. 32) which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 1 is objected to because of the following informality:
Claim 1 at step (d), part (ii), line 3: “wherein the exposing results in release of the extended nucleic acid capture probe of step (c) from the array” should be replaced with “wherein the exposing releases the extended nucleic acid capture probe of step (c) from the array.”
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 9, 11-17 and 21-23 and 31 are rejected are rejected under 35 U.S.C. 103 as being unpatentable over Godron et al. (WO 2020/120442 A2; published 6/18/2020).
Regarding claim 1, Godron teaches methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed and in some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3'-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3' hydroxyl, and further this invention has applications in single-cell cDNA library construction and analysis (Abstract). Godron also teaches that in some embodiments, the invention is directed to methods of generating cell- specific cDNA libraries each with cell-specific oligonucleotide barcodes comprising the steps of: (a) capturing mRNA of a single cell by hybridizing the mRNA to capture oligonucleotides attached to a bead, wherein the capture oligonucleotides are complementary to segments of the mRNA and wherein the capture oligonucleotides are attached to the bead by 5-ends and have free 3’ -hydroxyls; (b) extending 3’ -ends of the capture oligonucleotides with a reverse transcriptase using the captured mRNAs as templates to form cell-specific cDNA libraries; (c) synthesizing a unique cell-specific oligonucleotide barcode on each cDNA of a bead by template-free enzymatic synthesis. In some embodiments, the unique cell- or bead-specific barcode is a random sequence oligonucleotide and the step of synthesizing such barcode is carried out by a“split and mix” procedure with template-free enzymatic synthesis and further in some embodiments, the invention is directed to methods of extending one or more native polynucleotides with a predetermined nucleotide sequence, comprising: providing one or more native polynucleotides in a reaction mixture under TdT reaction conditions, the native polynucleotides having free 3’ -hydroxyls; and extending the one or more native polynucleotide with a predetermined sequence of nucleotides by repeated cycles of the steps (Paragraphs 7-8).
Godron further teaches that in some embodiments, this application of the invention may be carried out with the following steps: (a) providing an array comprising a uniform coating of capture probes each comprising a capture segment; (b) contacting a tissue sample with the array and allowing the nucleic acid of the tissue sample to interact with the capture domain of the capture probe so that the nucleic acid is captured; (c) treating the tissue sample to identify different regions of the tissue sample; (d) generating a nucleic acid molecule from the nucleic acid that interacts with the capture domain; (e) enzymatically synthesizing position tags onto the nucleic acid molecules; (f) determining the region that is associated with the nucleic acid that interacts with the capture domain; and (e) correlating the determined regions to the cDNAs; and further in some embodiments, the nucleic acid molecules from the tissue sample is RNA; and further in some other embodiments, the nucleic acid molecules from the tissue sample may be genomic DNA; and further in other embodiments, the nucleic acid molecules from the tissue sample may be mRNA (Paragraph 70, lines 1-10). Also, Godron teaches that similar to adding unique cellular tags in the case of beads described above, the invention also may be used to attach unique position tags in the case of spatial patterns of gene expression in a tissue slice disposed on a planar surface, as illustrated in Figs. IF and procedures for placing tissue slices on a planar array of oligonucleotides, identifying and imaging tissue features (such as cell boundaries), permeabilizing cells of tissues, implementing reverse transcriptase reactions to produce a cDNA library attached to a planar array (Paragraph 69, lines 1-5).
Additionally, Godron teaches that the invention process is illustrated in Figs. 1B-1C which show an embodiment where cDNAs have an oligonucleotide synthesized onto their 3’ ends and such cDNAs may be obtained, for example, from single cells isolated in reaction chambers. Cell-specific (or chamber- specific) oligonucleotides may be synthesized on such cDNAs, where afterwards, the contents of such reaction chambers may be combined, and the oligonucleotide-cDNA conjugates analyzed by large-scale sequence analysis to provide, for example, a single-cell transcriptome analysis of a population of cells, or transcriptome analysis of a group of cells from the same reactor that have been exposed to the same conditions and primer (102) having 3’-polyT portion (104) is annealed to poly A region of messenger RNAs (mRNAs) (100) and extended (108) with a reverse transcriptase using conventional protocols to form cDNA (113)(SEQ ID NO: 16) where primer (102) also has portion (106) which provides a means for attaching the resulting extension products (i.e. the cDNAs) to a solid support (Paragraph 31, lines 1-10). Further, Godron teaches that a wide variety of such attachment means are available including, but not limited to, a 5’ oligonucleotide tail which may be anneal to a complementary strand attached to a solid support, a member of a click chemistry reaction pair which may be reacted with a complementary member attached to a solid support to form a covalent bond, a member of a non-nucleic acid binding pair which may form a complex with a complementary member attached to a solid phase support to form a non-covalent bond, an example of the latter being biotin and streptavidin and returning to Fig. IB, two of the above attachment modes are illustrated with solid support (120a) which has complementary oligonucleotide (112) attached by its 3’ end and which captures cDNA (113) by forming a hybrid with portion (106) of extended primer (102), and with alternative support (120b) which has a member of a binding pair (114a)(such as, streptavidin) attached and which captures its complementary member (such as, biotin), where the member of a binding pair attached to the cDNA does not require the presence of oligonucleotide tail (106); however, in some embodiments, such oligonucleotide (106) may include nucleotides or nucleotide sequences which may be employed for cleaving finished product from the solid supports, e.g. presence of a uracil for cleavage by USER treatment, or the recognition sequence of a restriction endonuclease, such as a nickase (Paragraph 31, lines 10-20).
Specifically, Godron teaches that synthesizing oligonucleotides on living cells requires mild deblocking, or deprotection, conditions, that is, conditions that do not disrupt cellular membranes, denature proteins, interfere with key cellular functions, or the like, and in some embodiments, deprotection conditions are within a range of physiological conditions compatible with cell survival. In such embodiments, enzymatic deprotection is desirable because it may be carried out under physiological conditions, and further in some embodiments specific enzymatically removable blocking groups are associated with specific enzymes for their removal where for example, ester- or acyl- based blocking groups may be removed with an esterase, such as acetylesterase, or like enzyme, and a phosphate blocking group may be removed with a 3’ phosphatase, such as T4 polynucleotide kinase and by way of example, 3’ -O-phosphates may be removed by treatment with as solution of 100 mM Tris-HCl (pH 6.5) 10 mM MgC 12 , 5 mM 2-mercaptoethanol, and one Unit T4 polynucleotide kinase and the reaction proceeds for one minute at a temperature of 37°C (Paragraph 47, lines 1-10).
Godron also teaches that in Fig. 3C, for some embodiments, barcoded cells (340) may be prepared for transcriptome analysis using a droplet-based microfluidic device (345), which encapsulates barcoded single cells into aqueous micelles and coalesces the cell-containing micelles with a series of micelles containing reagents for constructing cDNA libraries and alternatively, cell- containing micelles may be produced and reagents delivered to such micelles using non microfluidic methods such as disclosed in Abate et al, International patent publication WO2019/139650 and cells (340) with initiator-barcode conjugates (344) embedded in their cell surface membranes are disposed in chamber (343) in aqueous solution (342) which may have a pH, salt concentrations and other necessary ingredients to maintain the integrity of the cells (Paragraph 86, lines 1-10). Further, Godron teaches that Exemplary lysis conditions for use with PCA reactions are as follows: 1) cells in H20 at 96° C. for 15 min, followed by 15 min at 10° C.; 2) 200 mM KOH, 50 mM dithiotheitol, heat to 65° C. for 10 min; 3) for 4 pL protease-based lysis buffer: 1 pL of 17 pM SDS combined with 3 pL of 125 pg/mL proteinase K, followed by incubation at 37° C. for 60 min, then 95° C. for 15 min (to inactivate the proteinase K); 4) for 10 pL of a detergent-based lysis buffer: 2 pL H20, 2 pL 250 ng/pL poly A, 2 pL 10 mM EDTA, 2 pL 250 mM dithiothreitol, 2 pL 0.5% N- laurylsarcosin salt solution and single-cell analysis platforms, incubation times, lysis buffer and/or PCA reaction other components, their concentrations, reactions volumes and the like, are design choices that are optimized for particular applications by one of ordinary skill in the art. In one embodiment, an alkaline lysis buffer disclosed by Kim et al, Anal. Chem., 90: 1273- 1279 (2018) is employed. Such buffer comprises 20 mM NaOH, 60% (v/v) PeG-200, and 2% (v/v) Triton X-100, and may be neutralized by the buffering capacity of an RT-PCR reagents (Paragraph 87, lines 10-20).
Regarding claim 2, Godron teaches that the previously explained method includes depending on particular applications, the steps of deblocking and/or cleaving may include a variety of chemical or physical conditions, e.g. light, heat, pH, presence of specific reagents, such as enzymes, which are able to cleave a specified chemical bond, as well as guidance in selecting 3’ -O-blocking groups and corresponding de-blocking conditions (Paragraph 45, lines 1-5). Further, Godron teaches that synthesizing oligonucleotides on living cells requires mild deblocking, or deprotection, conditions, that is, conditions that do not disrupt cellular membranes, denature proteins, interfere with key cellular functions, or the like and in some embodiments, deprotection conditions are within a range of physiological conditions compatible with cell survival, and further in such embodiments, enzymatic deprotection is desirable because it may be carried out under physiological conditions and further in some embodiments specific enzymatically removable blocking groups are associated with specific enzymes for their removal where for example, ester- or acyl- based blocking groups may be removed with an esterase, such as acetylesterase, or like enzyme, and a phosphate blocking group may be removed with a 3’ phosphatase, such as T4 polynucleotide kinase (Paragraph 47, lines 1-5). Further Godron teaches that by way of example, 3’ -O-phosphates may be removed by treatment with as solution of 100 mM Tris-HCl (pH 6.5) 10 mM MgC 12 , 5 mM 2-mercaptoethanol, and one Unit T4 polynucleotide kinase and the reaction proceeds for one minute at a temperature of 37°C (Paragraph 47, lines 5-10).
Regarding claim 3, Godron teaches that the previously described method includes similar to adding unique cellular tags in the case of beads described above, the invention also may be used to attach unique position tags in the case of spatial patterns of gene expression in a tissue slice disposed on a planar surface, as illustrated in Figs. IF and procedures for placing tissue slices on a planar array of oligonucleotides, identifying and imaging tissue features (such as cell boundaries), permeablizing cells of tissues, implementing reverse transcriptase reactions to produce a cDNA library attached to a planar array are disclosed in Stahl et al, Science, 353 : 78-82 (2016); and Frisen et al, U.S. patents 9593365 and 10030261 ; and like references, which are incorporated herein by reference and briefly, referring to Fig. IF, planar array (164) is provided with a uniform coating of oligonucleotides (180), with a controlled density, attached by their 5’ ends, wherein the oligonucleotides (shown in magnified view (165)) comprise segment (166), such as a primer binding site, for later amplification and manipulation of a cDNA, optional segment (167) comprising a molecular tag (sometimes referred to as a “unique molecular identifier” or UMI) which facilitates quantification of cDNA molecules even after amplification, and segment (168), such as a polyT segment, which permits capture of mRNA released from cells. The UMI (167) may comprise a random nucleotide segment (Paragraph 69, lines 1-10). Additionally, Godron teaches that oligonucleotides (180) may be made in bulk using conventional techniques and applied to the surface of planar array (164) in a single step and different kinds of oligonucleotides, for example, oligonucleotides with different position tags are not required. Segment (167) may also include a cleavable linker or cleavable nucleotide for releasing cDNAs for analysis, such as, by sequencing and onto array (164) is disposed a slice or thin layer (181) (e.g. 100-1000 pm thick) of tissue, which it is then treated (169) (i) to identify features, such as cells or sub-tissues, of interest and to record and/or correlate such information to locations on planar array (164), and (ii) to permeabilized cells in the tissue so that mRNA is released and allowed to diffuse to and be captured by oligonucleotides (180), where the image information is used to define regions on array (164) within which common position tags are synthesized on cDNAs and treatments may include staining with tissue-specific or biomolecule-specific compounds or dyes (Paragraph 69, lines 10-15). Godron further teaches that the position tags allow cDNAs to be harvested and sequenced in bulk, yet be related to specific regions by their position tags and after the above steps (i) and (ii), reagents for a reverse transcriptase reaction are applied in order to synthesize cDNAs (171) using captured mRNAs (170) as templates to produce a spatial cDNA library array, where tissue slice (181) is then removed leaving array (164) with a pattern of different cDNAs attached to its surface and the different cDNAs at the different positions may be identified and quantified by attaching position tags to samples of cDNAs from a plurality of locations by inkjet delivery of synthesis reagents for the tags, which is illustrated in Fig. IE by the superposition of synthesis locations (182) on cDNA pattern (175) (Paragraph 69, lines 15-25).
Regarding claim 4, Godron teaches that the previously described method includes that the invention process is illustrated in Figs. 1B-1C which show an embodiment where cDNAs have an oligonucleotide synthesized onto their 3’ ends and such cDNAs may be obtained, for example, from single cells isolated in reaction chambers. Cell-specific (or chamber- specific) oligonucleotides may be synthesized on such cDNAs, where afterwards, the contents of such reaction chambers may be combined, and the oligonucleotide-cDNA conjugates analyzed by large-scale sequence analysis to provide, for example, a single-cell transcriptome analysis of a population of cells, or transcriptome analysis of a group of cells from the same reactor that have been exposed to the same conditions and primer (102) having 3’-polyT portion (104) is annealed to poly A region of messenger RNAs (mRNAs) (100) and extended (108) with a reverse transcriptase using conventional protocols to form cDNA (113)(SEQ ID NO: 16) where primer (102) also has portion (106) which provides a means for attaching the resulting extension products (i.e. the cDNAs) to a solid support (Paragraph 31, lines 1-10). Further, Godron teaches that a wide variety of such attachment means are available including, but not limited to, a 5’ oligonucleotide tail which may be anneal to a complementary strand attached to a solid support, a member of a click chemistry reaction pair which may be reacted with a complementary member attached to a solid support to form a covalent bond, a member of a non-nucleic acid binding pair which may form a complex with a complementary member attached to a solid phase support to form a non-covalent bond, an example of the latter being biotin and streptavidin and returning to Fig. IB, two of the above attachment modes are illustrated with solid support (120a) which has complementary oligonucleotide (112) attached by its 3’ end and which captures cDNA (113) by forming a hybrid with portion (106) of extended primer (102), and with alternative support (120b) which has a member of a binding pair (114a)(such as, streptavidin) attached and which captures its complementary member (such as, biotin), where the member of a binding pair attached to the cDNA does not require the presence of oligonucleotide tail (106); however, in some embodiments, such oligonucleotide (106) may include nucleotides or nucleotide sequences which may be employed for cleaving finished product from the solid supports, e.g. presence of a uracil for cleavage by USER treatment, or the recognition sequence of a restriction endonuclease, such as a nickase (Paragraph 31, lines 10-20).
Regarding claims 9, 11-15, Godron also teaches that in Fig. 3C, for some embodiments, barcoded cells (340) may be prepared for transcriptome analysis using a droplet-based microfluidic device (345), which encapsulates barcoded single cells into aqueous micelles and coalesces the cell-containing micelles with a series of micelles containing reagents for constructing cDNA libraries and alternatively, cell- containing micelles may be produced and reagents delivered to such micelles using non microfluidic methods such as disclosed in Abate et al, International patent publication WO2019/139650 and cells (340) with initiator-barcode conjugates (344) embedded in their cell surface membranes are disposed in chamber (343) in aqueous solution (342) which may have a pH, salt concentrations and other necessary ingredients to maintain the integrity of the cells (Paragraph 86, lines 1-10). Further, Godron teaches that Exemplary lysis conditions for use with PCA reactions are as follows: 1) cells in H20 at 96° C. for 15 min, followed by 15 min at 10° C.; 2) 200 mM KOH, 50 mM dithiotheitol, heat to 65° C. for 10 min; 3) for 4 pL protease-based lysis buffer: 1 pL of 17 pM SDS combined with 3 pL of 125 pg/mL proteinase K, followed by incubation at 37° C. for 60 min, then 95° C. for 15 min (to inactivate the proteinase K); 4) for 10 pL of a detergent-based lysis buffer: 2 pL H20, 2 pL 250 ng/pL poly A, 2 pL 10 mM EDTA, 2 pL 250 mM dithiothreitol, 2 pL 0.5% N- laurylsarcosin salt solution and single-cell analysis platforms, incubation times, lysis buffer and/or PCA reaction other components, their concentrations, reactions volumes and the like, are design choices that are optimized for particular applications by one of ordinary skill in the art and in one embodiment, an alkaline lysis buffer disclosed by Kim et al, Anal. Chem., 90: 1273- 1279 (2018) is employed. Such buffer comprises 20 mM NaOH, 60% (v/v) PeG-200, and 2% (v/v) Triton X-100, and may be neutralized by the buffering capacity of an RT-PCR reagents (Paragraph 87, lines 10-20).
Regarding claims 16-17, 21, Godron teaches that the previously described method includes similar to adding unique cellular tags in the case of beads described above, the invention also may be used to attach unique position tags in the case of spatial patterns of gene expression in a tissue slice disposed on a planar surface, as illustrated in Figs. IF and procedures for placing tissue slices on a planar array of oligonucleotides, identifying and imaging tissue features (such as cell boundaries), permeabilizing cells of tissues, implementing reverse transcriptase reactions to produce a cDNA library attached to a planar array are disclosed in Stahl et al, Science, 353 : 78-82 (2016); and Frisen et al, U.S. patents 9593365 and 10030261 ; and like references, which are incorporated herein by reference and briefly, referring to Fig. IF, planar array (164) is provided with a uniform coating of oligonucleotides (180), with a controlled density, attached by their 5’ ends, wherein the oligonucleotides (shown in magnified view (165)) comprise segment (166), such as a primer binding site, for later amplification and manipulation of a cDNA, optional segment (167) comprising a molecular tag (sometimes referred to as a “unique molecular identifier” or UMI) which facilitates quantification of cDNA molecules even after amplification, and segment (168), such as a polyT segment, which permits capture of mRNA released from cells. The UMI (167) may comprise a random nucleotide segment (Paragraph 69, lines 1-10). Additionally, Godron teaches that oligonucleotides (180) may be made in bulk using conventional techniques and applied to the surface of planar array (164) in a single step and different kinds of oligonucleotides, for example, oligonucleotides with different position tags are not required. Segment (167) may also include a cleavable linker or cleavable nucleotide for releasing cDNAs for analysis, such as, by sequencing and onto array (164) is disposed a slice or thin layer (181) (e.g. 100-1000 pm thick) of tissue, which it is then treated (169) (i) to identify features, such as cells or sub-tissues, of interest and to record and/or correlate such information to locations on planar array (164), and (ii) to permeabilized cells in the tissue so that mRNA is released and allowed to diffuse to and be captured by oligonucleotides (180), where the image information is used to define regions on array (164) within which common position tags are synthesized on cDNAs and treatments may include staining with tissue-specific or biomolecule-specific compounds or dyes (Paragraph 69, lines 10-15). Godron further teaches that the position tags allow cDNAs to be harvested and sequenced in bulk, yet be related to specific regions by their position tags and after the above steps (i) and (ii), reagents for a reverse transcriptase reaction are applied in order to synthesize cDNAs (171) using captured mRNAs (170) as templates to produce a spatial cDNA library array, where tissue slice (181) is then removed leaving array (164) with a pattern of different cDNAs attached to its surface and the different cDNAs at the different positions may be identified and quantified by attaching position tags to samples of cDNAs from a plurality of locations by inkjet delivery of synthesis reagents for the tags, which is illustrated in Fig. IE by the superposition of synthesis locations (182) on cDNA pattern (175) (Paragraph 69, lines 15-25).
Regarding claims 21-23, Godron teaches that in some embodiments, the invention is directed to a method of generating a cell- specific cDNA library with cell-specific oligonucleotide barcodes comprising the steps of: (a) synthesizing a unique oligonucleotide barcode on a cell surface membrane of each cell in a population of cells to form a population of barcoded cells; (b) isolating each barcoded cell in a reactor; (c) lysing barcoded cells in each reactor; (d) performing reverse-transcriptase polymerase chain reaction (RT-PCR) in each reactor to produce a cDNA library with cell- specific oligonucleotide barcodes and further in some embodiments, the step of synthesizing is carried out by a template-free enzymatic synthesis method of the invention. In some embodiments, the RT-PCR reaction includes attaching barcodes to cDNAs by a polymerase cycling amplification reaction (Paragraph 6, lines 1-10).
Regarding claim 31, Godron teaches methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed and in some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3'-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3' hydroxyl, and further this invention has applications in single-cell cDNA library construction and analysis (Abstract). Godron also teaches that in some embodiments, the invention is directed to methods of generating cell- specific cDNA libraries each with cell-specific oligonucleotide barcodes comprising the steps of: (a) capturing mRNA of a single cell by hybridizing the mRNA to capture oligonucleotides attached to a bead, wherein the capture oligonucleotides are complementary to segments of the mRNA and wherein the capture oligonucleotides are attached to the bead by 5-ends and have free 3’ -hydroxyls; (b) extending 3’ -ends of the capture oligonucleotides with a reverse transcriptase using the captured mRNAs as templates to form cell-specific cDNA libraries; (c) synthesizing a unique cell-specific oligonucleotide barcode on each cDNA of a bead by template-free enzymatic synthesis. In some embodiments, the unique cell- or bead-specific barcode is a random sequence oligonucleotide and the step of synthesizing such barcode is carried out by a“split and mix” procedure with template-free enzymatic synthesis and further in some embodiments, the invention is directed to methods of extending one or more native polynucleotides with a predetermined nucleotide sequence, comprising: providing one or more native polynucleotides in a reaction mixture under TdT reaction conditions, the native polynucleotides having free 3’ -hydroxyls; and extending the one or more native polynucleotide with a predetermined sequence of nucleotides by repeated cycles of the steps (Paragraphs 7-8). Further, Godron teaches, as illustrated in Fig. 1G, spatial distributions of proteins in a tissue sample may be identified by using antibodies with releasable oligonucleotide barcodes that contain a polyA region and an antibody identification region, that is, an antibody barcode that identifies the protein that the antibody is specific for. In some embodiments, antibodies may carry two tags; an antibody barcode as described above and a fluorescent label which would assist in the optical analysis of the tissue and later correlation of antibody positions with tissue structures or protein distributions of interest and as above, planar array (1164) is provided with a uniform coating of oligonucleotides (1180) attached by their 5’ ends, wherein the oligonucleotides (shown in magnified view (1165)) optionally comprise segment (1166), such as a primer binding site, for later amplification and manipulation of an antibody barcode, optional segment (1167) comprising a molecular tag (sometimes referred to as a“ unique molecular identifier” or UMI) which facilitates quantification of antibody molecules even after amplification, and segment (1168), such as a polyT segment, which permits capture of antibody barcodes (1 183) (SEQ ID NO: 18) released from bound antibodies (Paragraph 71, lines 1-10).
Godron also teaches that Figs. 1D-1E illustrate how the above methods may be used with commercially available polyT beads to construct solid phase cDNA libraries, e.g. Dynabeads™ oligo(dT) magnetic beads, Bosnes et al, ThermoFisher Application Note (2017) where PolyT beads (150) are combined with cell extract or lysate containing polyA RNA (152) under conditions that permit hybridization of the polyT segments of the beads to the polyA segments of the RNAs, after which the hybridized polyT segments are extended in a reverse transcription reaction and after removal of the RNA template (156), a solid phase cDNA library (158) results, which may then be processed in accordance with the method of Figs IB and 1C (160) to synthesize barcodes, primer binding sites, or the like (162), to allow further analysis of the cDNAs where such barcodes may uniquely designate a particular sample, such as a patient sample, or as described further below, such barcodes may designate uniquely a single cell (Paragraph 34, lines 1-10).
Specifically, Godron teaches that an alternative application for single-cell transcriptome analysis is illustrated in Fig. 5. In this embodiment, cells (without barcodes)(502) are mixed with polyT beads (also without barcodes)(504) and disposed in an aqueous mixture in chamber (500) of microfluidics device (508) and as mentioned above, approaches not depending on microfluidics devices may also be applied, e.g. Abate et al, International patent publication WO2019/139650, where the aqueous mixture is forced through passage (506) into oil stream (512) at junction (510) so that aqueous droplets form, some of which (516) contain one cell (517a) and one bead (517b) and such droplets are then coalesced with droplets (518) containing cell lysis reagents at junction (520) to form droplet (522) in which cells are lysed releasing polyA RNA, which anneals to the polyT primers attached to beads (517b) and after appropriate incubation to release the desired cellular constituents, such as mRNA, the lysate-containing droplets are then coalesced at junction (530) with droplets (528) containing reverse transcriptase reagents, such as a reverse transcriptase, appropriate salts, and a buffer system that may counteract or alter conditions (e.g. high pH) imposed by the lysis reaction where then resulting droplets (531) are collected (532) and incubated so that polyA RNA anneals to polyT primers on beads (517b) and serves as a template for the reverse transcriptase extension of the polyT segments to form a single-cell cDNA library covalently attached to bead (517b) and beads (517b) may then be collected from the droplets and combined and subjected to“split and mix” synthesis to add a unique barcode and further sequences, such as primer binding sites, for subsequent manipulation, such as copying and preparation of high throughput sequencing, as described above (Paragraph 03, lines 1-20).
As showcased above, Godron teaches capture oligonucleotides attached to a solid support by their 5’ ends and having free 3’ hydroxyl groups, wherein the 3’ ends of the capture oligonucleotides are extended with a reverse transcriptase using captured mRNAs as templates to form cDNA. Thus, Gordon teaches formation of an extended, support-bound nucleic acid capture probe. Gordon does not teach or suggest using its disclosed alkaline and detergent conditions to release the extended nucleic acid capture probe from the array. However, Godron expressly teaches that its array-bound cDNAs are harvested/released for subsequent analysis; teaches nucleic acid processing conditions employing KOH, NaOH, SDS and Triton-X 100; teaches the use of Tris-HCl buffer in nucleic acid processing; and teaches spatial analysis of tissue sections on a planar array, as previously shown.
Therefore, it would have been obvious to one of ordinary skill in the art to employ Godron’s disclosed alkaline and detergent conditions as an alternative chemical means for releasing the extended nucleic acid capture probes from the array, to neutralize the resulting alkaline solution using a conventional buffer such as Godron’s disclosed Tris buffer, and to employ a fresh-frozen tissue section in the disclosed spatial transcriptomic workflow. One of ordinary skill would have been motivated to make these modifications to provide an alternative chemical means for recovering Godron’s array-bound cDNA for its expressly contemplated downstream amplification and sequencing, to restore the alkaline reaction mixture to a pH suitable for subsequent nucleic-acid processing, and to preserve RNA integrity for capture and reverse transcription. There would have been a reasonable expectation of success because Godron itself teaches the relevant alkaline/detergent reagents, Tris buffering conditions, array-bound cDNA workflow, and subsequent recovery and analysis of the cDNA, while fresh-frozen tissue was a conventional tissue preparation for RNA-based analysis. Selection and optimization of the disclosed reagents, concentrations, exposure conditions, buffer, and tissue preparation would therefore have involved routine optimization of known conditions for their intended purposes.
Conclusions
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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