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 .
Claim Status
Claim 23-32, 34, 36, 38, and 40-48 are pending and under examination.
Claims 1-22, 33, 35, 37, and 39 have been canceled.
Response to Amendment
Applicant’s amendments to the claims and remarks, received 01/14/2026, have overcome the 112(b) rejection(s) previously set forth in the Non-Final Office Action mailed on 07/14/2025.
Based on the amended claims and remarks, the prior art rejection over on Shema-Yaacoby has been modified to address the amended claims (see below).
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 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
Claim(s) 23-32, 34, 36, 38, 40-48 are rejected under 35 U.S.C. 103 as being unpatentable over Shema-Yaacoby et al. (WO 2017/034970 A1 where US 2019/0284603 is used as the corresponding document - hereinafter “Yaacoby”; already of record), in view of Rotem et al. (US 2015/0057163 - hereinafter “Rotem”; already of record) and further in view of Epstein et al. (US 2017/0343539 – hereinafter “Epstein”; already of record) , and further in view of Bibillo et al. (US 2019/0040454 – hereinafter “Bibillo”; already of record).
Regarding claim 23, Yaacoby discloses a method determining an epigenetic state of a biological element (Yaacoby discloses technologies to study the contribution of epigenetic mechanisms to cancer initiation and progression; [0017] including a method for screening drug targets and drug candidates based on changes in the combinatorial pattern of histone modifications; [0018]), comprising:
(a) providing a plurality of isolated first droplet that comprise: i) a biological element, wherein the biological element contains or is suspected of containing at least one nucleosome comprising one or more epigenetic features and a genomic region, ii) a lysis buffer, and iii) a nuclease (Yaacoby discloses the method comprises obtaining isolated chromatin fragments by separating single cells into droplets formed by an aqueous solution in oil emulsion, wherein each droplet comprises a single cell, a nuclease, and a lysis buffer; [0025, 0032]);
(d) providing a plurality of isolated second droplets that comprise a nucleic acid molecule comprising a barcode sequence, an adaptor, and a protecting function against unwanted ligation (Yaacoby discloses covalently linking an oligonucleotide sequence to the isolated chromatin fragments by introducing a second droplet comprising the oligonucleotide and a barcode; [0019, 0025, 0032]. The oligonucleotides comprise either a ssDNA, biotin, phosphoryl group, or a restriction enzyme site at the 3’ end or 5’ end; [0020, 0260]);
(e) processing the nucleic acid molecule of one or more isolated second droplets of the plurality of isolated second droplets by fusing an isolated first droplet of the plurality of isolated first droplets with an isolated second droplet of the plurality of isolated second droplets to identify said one or more epigenetic features, wherein processing comprises: (i) ligating the barcode sequence and the adaptor to an end of the genomic region of one or more nucleosomes to obtain one or more barcoded nucleosome sequences, and (ii) obtaining sequence information from one or more barcoded nucleosome sequences comprising the barcode sequence on the nucleosome sequence (Yaacoby; Introducing the oligonucleotide sequence may comprise fusing the second droplet with the first droplet; [0025]. The method is used in the study of epigenetics as discussed in “Example 10”. “Applicants can use the microfluidics to lyse single cells, digest their chromatin and index nucleosomes to originating cell. In this case, the nucleosomes are ligated to adaptors containing both barcode and biotin.”; fig. 5 – “Single nucleosome decoding”, [0017, 0205, 0253, 0255, 0258, 0260]); and
(f) using said one or more epigenetic features and said barcode sequence to determine barcode epigenetic state of the biological element of one or more of the plurality of isolated first droplets ([0017, 0205, 0254-0255]).
Yaacoby does not disclose (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets.
However, Rotem teaches the analogous art of a method for determining an epigenetic state of a biological element (Rotem; [0006]) comprising (a) providing a plurality of isolated first droplet comprising a biological element, a lysis buffer, and a nuclease (Rotem; fig. 2; [0027]), (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets (Rotem disclose cell lysis and chromatin digestions in encapsulated droplets where the cells are incubated for 10 min. at 4 degrees Celsius, 15 min. at 37 degrees Celsius, and put back at 4 degrees Celsius until the next step. Accordingly, each droplet undergoes steps (b) and (c) for a predetermined period of time to achieve synchronized collection, incubation, and enzymatic processing; fig. 2, [0094-0095]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to have modified the method of Yaacoby to comprise (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets, as taught by Rotem, because Rotem teaches synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets allows cell lysis and chromatin digestion in droplets using a microfluidic device where subsequent processing steps may be performed; fig. 2, [0094]. In addition, Yaacoby and Rotem both teach collecting the plurality of isolated first droplets in a collection tube equivalent to applicant’s collection tube disclosed in their printed publication (See fig. 1 “collection tube” and paragraph [0166] of applicant’s printed publication). Specifically, Yaacoby teach a collection tube for collecting the first type of droplets; fig. 5 “Pool and add unlabeled carrier chromatin”, [00279, 0300] and Rotem teach plates/tubes/containers/vials for collecting the first type of droplets where steps (b) and (c) are performed on each droplet [0054, 0078, 0097]. Accordingly, modified Yaacoby teach the claimed synchronized collecting, incubating, and processing of the plurality of isolated first droplets to thereby synchronously activate the nuclease. One of ordinary skill in the art would have expected this modification could have been performed with a reasonable expectation of success since Yaacoby and Rotem both teach a method for epigenetic sequencing using barcoded adaptors.
Modified Yaacoby does not teach one or more asymmetrically barcoded nucleosome sequences comprising the barcode sequence on only one end of the nucleosome sequence.
However, Epstein teaches the analogous art of labeling fragmented nucleosomal DNA with barcoded adapters, wherein the barcoded adapters may comprise DNA, RNA, nucleotide analogs or combinations thereof, and a protecting function comprising a three-carbon spacer, wherein the nucleosomal DNA is labeled on only a single free end (Epstein; [0055-0056]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, because Epstein teaches the barcoded adapter comprising the protecting function prevents self-ligation and concatemerization of the adapter at the 5’ end; [0056]. Thus, obtaining sequence information from one or more asymmetrically barcoded nucleosome sequences comprising the barcode sequence on only one end of the nucleosome sequence; [0007, 0055-0056]. One of ordinary skill in the art would have expected this modification could have been performed with a reasonable expectation of success since modified Yaacoby and Epstein both teach labeling nucleosomal DNA with adapters.
Modified Yaacoby does not teach wherein the protecting function against unwanted ligation is on a 3’-end of the nucleic acid molecule. However, Bibillo teach the analogous art of a method for detecting, diagnosing, and/or prognosing cancer in a subject using sequence information of a nucleic acid molecule (Bibillo; [0189]) wherein the nucleic acid molecule is modified to include the addition of a C3 spacer to the 3’ end of a polynucleotide (Bibillo; [0113]). It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, because Bibillo teach the C3 spacer on the 3’-end of the nucleic acid molecule protects the nucleic acid molecule from being mistaken as an RNA primer by functioning as an effective blocking agent against polymerase extension (Bibillo; [0113]).
Regarding claim 24, modified Yaacoby teach the method of claim 23 above, wherein the biological element is chosen from a single cell, a nucleus, and a nucleic acid-containing organelle (Yaacoby; [0025]).
Regarding claim 25, modified Yaacoby teach the method of claim 23 above, wherein said one or more epigenetic features comprise one or more DNA or protein modifications (Yaacoby; chromatin fragments refer to a mixture of DNA and proteins. See Example 10. [0025, 0032, 0251-0260]).
Regarding claim 26, modified Yaacoby teach the method of claim 23 above, wherein the one or more epigenetic features comprise a post-translational modification chosen from acetylation, amidation, deamidation, carboxylation, disulfide bond, formylation, glycosylation, hydroxylation, methylation, myristoylation, nitrosylation, phosphorylation, prenylation, ribosylation, sulphation, sumoylation, ubiquitination and derivatives thereof (Yaacoby; [0047]).
Regarding claim 27, modified Yaacoby teach the method of claim 23 above, wherein the one or more epigenetic features comprise the modification of the presence or absence of methylation of DNA in at least one gene (Yaacoby; [0047, 0091]).
Regarding claim 28, modified Yaacoby teach the method of claim 23 above, wherein the protecting function is a spacing element or a dideoxy-modified base (The modification of the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, has previously been discussed in claim 23 above. Bibillo teach the addition of 3’ C3 spacer to the 3’ end of a polynucleotide or dideoxy 3’ end; [0113]).
Regarding claim 29, modified Yaacoby teach the method of claim 23 above, wherein the nucleic acid molecule further comprises at least one cleavage site (Yaacoby discloses the method may comprise cleaving of the marker from the oligonucleotide sequence; [0021, 0052]).
Regarding claim 30, modified Yaacoby teach the method of claim 28 above, wherein the spacing element is a three carbon (C3) spacer (The modification of the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, has previously been discussed in claim 23 above. Bibillo teach the addition of 3’ C3 spacer to the 3’ end of a polynucleotide; [0113]).
Regarding claim 31, modified Yaacoby teach the method of claim 29 above, wherein the at least one cleavage site is a restriction site comprising a palindromic region (Yaacoby; [0132-0133, 0161]).
Regarding claim 32, modified Yaacoby discloses a method (Yaacoby; [0018]), comprising:
(i) determining an epigenetic state of a biological element from a subject in need thereof (Yaacoby discloses technologies and methods to study the contribution of epigenetic mechanisms to cancer initiation and progression; [0017-0018]),
(ii) diagnosing and/or prognosing drug resistance in the subject based on the epigenetic state (Yaacoby discloses the method comprises screening drug targets and drug candidates based on changes in the combinatorial pattern of histone modifications; [0018]), and
(iii) administering a therapeutic agent to the subject based on the diagnosis and/or prognosis of (ii) (Yaacoby; fig. 3D, [0074, 0225, 0258]),
wherein the epigenetic state of the biological element is determined using a method comprising:
(a) providing a plurality of isolated first droplets that comprise: i) a biological element, wherein the biological element contains or is suspected of containing at least one nucleosome comprising one or more epigenetic features and a genomic region, ii) a lysis buffer, and iii) a nuclease (Yaacoby discloses the method comprises obtaining isolated chromatin fragments by separating single cells into droplets formed by an aqueous solution in oil emulsion, wherein each droplet comprises a single cell, a nuclease, and a lysis buffer; [0025, 0032]);
(d) providing a plurality of isolated second droplets that comprise a nucleic acid molecule comprising a barcode sequence, an adaptor, and a protecting element against unwanted ligation (Yaacoby discloses covalently linking an oligonucleotide sequence to the isolated chromatin fragments by introducing a second droplet comprising the oligonucleotide and a barcode; [0019, 0025, 0032]. The oligonucleotides comprise either a ssDNA, biotin, phosphoryl group, or a restriction enzyme site at the 3’ end or 5’ end; [0020, 0260]);
(e) processing the nucleic acid molecule of one or more isolated second droplets of the plurality of isolated second droplets by fusing an isolated first droplet of the plurality of isolated first droplets with an isolated second droplet of the plurality of isolated second droplets to identify the one or more epigenetic features, wherein processing comprises: (i) ligating the barcode sequence and adaptor to an end of the genomic region of one or more nucleosomes to obtain one or more barcoded nucleosome sequences; and (ii) obtaining sequence information from one or more barcoded nucleosome sequences comprising the barcode sequence on the nucleosome sequence (Yaacoby; Introducing the oligonucleotide sequence may comprise fusing the second droplet with the first droplet; [0025]. The method is used in the study of epigenetics as discussed in “Example 10”. “Applicants can use the microfluidics to lyse single cells, digest their chromatin and index nucleosomes to originating cell. In this case, the nucleosomes are ligated to adaptors containing both barcode and biotin.”; fig. 5 – “Single nucleosome decoding”, [0017, 0205, 0253, 0255, 0258, 0260]); and
(f) using said one or more epigenetic features and said barcode sequence to determine the epigenetic state of the biological element of one or more of the plurality of isolated first droplets ([0017, 0205, 0254-0255]).
Yaacoby does not disclose (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplet under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets.
However, Rotem teaches the analogous art of a method for determining an epigenetic state of a biological element (Rotem; [0006]) comprising (a) providing a plurality of isolated first droplet comprising a biological element, a lysis buffer, and a nuclease (Rotem; fig. 2; [0027]), (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets (Rotem disclose cell lysis and chromatin digestions in encapsulated droplets where the cells are incubated for 10 min. at 4 degrees Celsius, 15 min. at 37 degrees Celsius, and put back at 4 degrees Celsius until the next step. Accordingly, each droplet undergoes steps (b) and (c) for a predetermined period of time to achieve synchronized collection, incubation, and enzymatic processing; fig. 2, [0094-0095]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to have modified the method of Yaacoby to comprise (b) synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of isolated first droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and (c) incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets, as taught by Rotem, because Rotem teaches synchronizing enzymatic activity of the nuclease in the plurality of isolated first droplets by collecting the plurality of droplets under conditions that temporarily inactivate said nuclease, wherein the plurality of isolated first droplets is collected at a temperature of -20°C to 10°C and incubating the collected plurality of isolated first droplets at a temperature of 20°C to 40°C to thereby synchronously activate the nuclease in the plurality of isolated first droplets allows cell lysis and chromatin digestion in droplets using a microfluidic device where subsequent processing steps may be performed; fig. 2, [0094]. In addition, Yaacoby and Rotem both teach collecting the plurality of isolated first droplets in a collection tube equivalent to applicant’s collection tube disclosed in their printed publication (See fig. 1 “collection tube” and paragraph [0166] of applicant’s printed publication). Specifically, Yaacoby teach a collection tube for collecting the first type of droplets; fig. 5 “Pool and add unlabeled carrier chromatin”, [00279, 0300] and Rotem teach plates/tubes/containers/vials for collecting the first type of droplets where steps (b) and (c) are performed on each droplet [0054, 0078, 0097]. Accordingly, modified Yaacoby teach the claimed synchronized collecting, incubating, and processing of the plurality of isolated first droplets to thereby synchronously activate the nuclease. One of ordinary skill in the art would have expected this modification could have been performed with a reasonable expectation of success since Yaacoby and Rotem both teach a method for epigenetic sequencing using barcoded adaptors.
Modified Yaacoby does not teach one or more asymmetrically barcoded nucleosome sequences comprising the barcode sequence on only one end of the nucleosome sequence.
However, Epstein teaches the analogous art of labeling fragmented nucleosomal DNA with barcoded adapters, wherein the barcoded adapters may comprise DNA, RNA, nucleotide analogs or combinations thereof, and a protecting function comprising a three-carbon spacer, wherein the nucleosomal DNA is labeled on only a single free end (Epstein; [0055-0056]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, because Epstein teaches the barcoded adapter comprising the protecting function prevents self-ligation and concatemerization of the adapter at the 5’ end; [0056]. Thus, obtaining sequence information from one or more asymmetrically barcoded nucleosome sequences comprising the barcode sequence on only one end of the nucleosome sequence; [0007, 0055-0056]. One of ordinary skill in the art would have expected this modification could have been performed with a reasonable expectation of success since modified Yaacoby and Epstein both teach labeling nucleosomal DNA with adapters.
Modified Yaacoby does not teach wherein the protecting element against unwanted ligation is on a 3’-end of the nucleic acid molecule. However, Bibillo teach the analogous art of a method for detecting, diagnosing, and/or prognosing cancer in a subject using sequence information of a nucleic acid molecule (Bibillo; [0189]) wherein the nucleic acid molecule is modified to include the addition of a C3 spacer to the 3’ end of a polynucleotide (Bibillo; [0113]). It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the nucleic acid molecule and protecting element of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, because Bibillo teach the C3 spacer on the 3’-end of the nucleic acid molecule protects the nucleic acid molecule from being mistaken as an RNA primer by functioning as an effective blocking agent against polymerase extension (Bibillo; [0113]).
Regarding claim 34, modified Yaacoby teach the method of claim 32 above, wherein the subject is or is suspected to be in a disease state chosen from cancer, an infectious disease, an autoimmune disease, a metabolic disease, an inflammation disease, genetic diseases, and non-genetic diseases (Yaacoby; [0055])
Regarding claim 36, modified Yaacoby teach the method of claim 32 above, wherein the subject is treated with a therapeutic agent chosen from a chemotherapeutic agent, a chemical drug, and biological drug (Yaacoby; [0018, 0074, 0084, 0225, 0258]).
Regarding claim 38, modified Yaacoby teach the method of claim 32 above, wherein the diagnosing and/or prognosing of drug resistance is performed before, concurrent, or after the subject is treated with a first therapy (Yaacoby; [0055, 0199]).
Regarding claim 40, modified Yaacoby teach the method of claim 32 above, wherein the epigenetic state of the biological element comprises loss of one or more chromatin marks for genes that promote drug resistance (Yaacoby; fig. 2(G) demonstrates preferential loss of H3K27me3 from bivalent nucleosomes; [0073]).
Regarding claim 41, modified Yaacoby teach the method of claim 40 above, wherein the epigenetic state comprises the loss of one or more chromatin marks of H3K4me3 or H3K27me3 (Yaacoby; [0056, 0073]).
Regarding claim 42, modified Yaacoby teach the method of claim 23 above, further comprising performing a linear amplification of one or more asymmetrically barcoded nucleosome sequences comprising the barcode sequence on only one end of the nucleosome sequence (The modification of the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, has previously been discussed in claim 13 above. Yaacoby further teaches linear amplification; [0253]).
Regarding claim 43, modified Yaacoby teach the method of claim 23 above, further comprising obtaining sequence information from one or more symmetrically barcoded nucleosome sequences comprising a barcode sequence on both ends of the nucleosome sequence (The modification of the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, has previously been discussed in claim 23 above. Epstein teaches obtaining sequence information from one or more symmetrically or asymmetrically barcoded nucleosome sequences comprising a barcode sequence on one or both ends of the nucleosome sequence; [0007, 0055-0057]).
Regarding claim 44, modified Yaacoby teach the method of claim 23 above, wherein the nucleic acid molecule comprises the protecting function against unwanted ligation only on the 3’-end of the nucleic acid molecule (The modification of the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, and the modification of the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, have previously been discussed in claim 23 above. Bibillo teach the addition of 3’ C3 spacer only on the 3’ end of a polynucleotide; [0113]).
Regarding claim 45, modified Yaacoby teach the method of claim 23 above, wherein the nucleic acid molecule comprises (i) the protecting function against unwanted ligation on the 3’-end, and (ii) a 5’-end capable of ligation (The modification of the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, and the modification of the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, have previously been discussed in claim 23 above. Epstein teaches modifications to the adapter may be made to the first, second, third, and/or fourth terminal bases of the adaptor on the 3’ or 5’ end of the sense or antisense strand; [0056-0057]).
Regarding claim 46, modified Yaacoby teach the method of claim 23 above, wherein the nucleic acid molecule is a double-stranded nucleic acid molecule comprising a first strand and a second strand, wherein the first strand comprises the protecting function against unwanted ligation on its 3’-end and a 5’end capable of ligation, and wherein the second strand comprises a 3’-end capable of ligation (The modification of the nucleic acid molecule of modified Yaacoby with the barcoded adapter comprising DNA, RNA, and/or nucleotide analogs and a protecting function, as taught by Epstein, and the modification of the nucleic acid molecule and protecting function of modified Yaacoby with the C3 spacer on the 3’-end of the nucleic acid molecule, as in Bibillo, have previously been discussed in claim 23 above. Epstein teaches modifications to the adapter may be made to the first, second, third, and/or fourth terminal bases of the adaptor on the 3’ or 5’ end of the sense or antisense strand; [0056-0057]).
Regarding claim 47, modified Yaacoby teach the method of claim 46 above, wherein the second strand further comprises a linker providing releasable conjugation to a solid support on its 5’-end (Yaacoby; figs. 5 & 16).
Regarding claim 48, modified Yaacoby teach the method of claim 47 above, wherein the nucleic acid molecule is releasably conjugated to a solid support via the linker prior to the processing step and is released from the solid support prior to the ligating step (Yaacoby; figs. 5 & 16, [0231]).
Response to Arguments
Applicant’s arguments, filed 01/14/2026, have been fully considered but were not found persuasive by the examiner.
Applicant argues, see pages 7-9 of their remarks, that the cited references do not teach step (b), step (c), and step (e) are carried out in a synchronized manner. Applicant argues that paragraphs [0042, 0062, 0083, 0085] of their printed publication indicate that the feature of inactivation of MNase activity in the present application is not only a simple temporary inactivation, but rather, the inactivation is synchronized, to achieve equal treatment and incubation times for all droplets to reduce inter-droplet variability.
The examiner respectfully disagrees with applicant’s argument that the prior art does not teach step (b), step (c), and step (e) are carried out in a synchronized manner. Specifically, Rotem disclose cell lysis and chromatin digestions in encapsulated droplets where the cells are incubated for 10 min. at 4 degrees Celsius, 15 min. at 37 degrees Celsius, and put back at 4 degrees Celsius until the next step. Accordingly, each droplet undergoes steps (b) and (c) for a predetermined period of time to achieve synchronized collection, incubation, and processing; fig. 2, [0094-0095]. In addition, Yaacoby and Rotem both teach collecting the plurality of isolated first droplets in a collection tube equivalent to applicant’s collection tube disclosed in their printed publication (See fig. 1 “collection tube” and paragraph [0166] of applicant’s printed publication). Specifically, Yaacoby teach a collection tube for collecting the first type of droplets; fig. 5 “Pool and add unlabeled carrier chromatin”, [00279, 0300] and Rotem teach plates/tubes/containers/vials for collecting the first type of droplets where steps (b) and (c) are performed on each droplet synchronously [0054, 0078, 0097]. Accordingly, modified Yaacoby teach the claimed synchronized collecting, incubating, and processing of the plurality of isolated first droplets to thereby synchronously activate the nuclease.
Citations to art
In the above citations to documents in the art, an effort has been made to specifically cite representative passages, however rejections are in reference to the entirety of each document relied upon. Other passages, not specifically cited, may apply as well.
Conclusion
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CURTIS A THOMPSON whose telephone number is (571)272-0648. The examiner can normally be reached on M-F: 7:00 a.m. - 5:00 p.m..
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
E-mail communication Authorization
Per updated USPTO Internet usage policies, Applicant and/or applicant’s representative is encouraged to authorize the USPTO examiner to discuss any subject matter concerning the above application via Internet e-mail communications. See MPEP 502.03. To approve such communications, Applicant must provide written authorization for e-mail communication by submitting the following statement via EFS Web (using PTO/SB/439) or Central Fax (571-273-8300):
Recognizing that Internet communications are not secure, I hereby authorize the USPTO to communicate with the undersigned and practitioners in accordance with 37 CFR 1.33 and 37 CFR 1.34 concerning any subject matter of this application by video conferencing, instant messaging, or electronic mail. I understand that a copy of these communications will be made of record in the application file.
Written authorizations submitted to the Examiner via e-mail are NOT proper. Written authorizations must be submitted via EFS-Web (using PTO/SB/439) or Central Fax (571-273-8300). A paper copy of e-mail correspondence will be placed in the patent application when appropriate. E-mails from the USPTO are for the sole use of the intended recipient, and may contain information subject to the confidentiality requirement set forth in 35 USC § 122. See also MPEP 502.03.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Charles Capozzi can be reached at 571-270-3638. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/C.A.T./Examiner, Art Unit 1798
/BENJAMIN R WHATLEY/Primary Examiner, Art Unit 1798