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 .
Status of The Claims
Claims 1-2, 4-6, and 11-24 are pending and are under examination.
Claim 3 is canceled.
Applicant’s election without traverse of Group I, which includes Claims 1-2, 4-6, and 11-24 in the reply filed on 04/10/2026 is acknowledged.
Claim 3 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected method for mediated pulled down library splitting workflow, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 04/20/2026.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 and 2 recites “A method for obtaining gene expression information for a single nucleus”, but later steps recite “one or more nuclei”. The scope of the invention is unclear whether the claimed method is limited to single nucleus or encompasses multiple nucleus.
Claim 1(g), (h) recites “the DNA” lacks proper antecedent basis. The claim previously recites “genomic DNA fragments”, but does not clearly define “the DNA”. It is unclear whether “the DNA” refers to genomic DNA fragments in step c, tagged genomic DNA fragments in step e, or other DNA species.
Claim 1(j)(ii) recites “the DNA end” lacks proper antecedent basis. The claim does not identify which DNA end is being contacted with the sequencing adaptor and ligase.
Claim 2(g) recites “the DNA” lacks proper antecedent basis. The claim previously recites “genomic DNA fragments”, but does not clearly define “the DNA”. It is unclear whether “the DNA” refers to genomic DNA fragments in step c, tagged genomic DNA fragments in step e, or other DNA species.
Claim 2(h) recites “the cDNA” lacks proper antecedent basis. The claim does not clearly define “the DNA”. It is unclear whether “the cDNA” refers to tagged genomic DNA fragments in step d, e, or other cDNA species.
Claim 2(j)(ii) recites “the cDNA end” lacks proper antecedent basis. The claim does not identify which DNA end is being contacted with the sequencing adaptor and ligase.
Claim 1 and 2 recites “a restriction an endonuclease” is grammatically unclear. It is unclear if the claims intended to recite “a restriction endonuclease” or “an endonuclease”.
Claims 4-6, and 11-23 depend from claims 1, Claims 24 depend from claims 2 and are therefore similarly rejected.
Claim 23 recites “the pooling”, “the dividing” and “the contacting” lacks proper antecedent basis. Claim 21 recites multiple rounds of pooling, diving, and contacting steps, and claim 23 does not specify which of those steps are repeated.
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.
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.
Zhu et al., Kaya-Okur et al. and Lundin et al.
Claim(s) 1-2, 5-6, 11-21, and 23-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. (Nat Struct Mol Biol 26, 1063–1070 (2019), disclosed in IDS )in view of Kaya-Okur et al. (Nat Commun 10, 1930 (2019), disclosed in IDS), Lundin et al. (PLoS One. 2015;10(1):e0117059), and Fernandez et al. (US9938524B2, published April 23rd 2015)
Regarding claim 1, Zhu discloses a method for obtaining gene expression information for a single nucleus, the method comprising:
a. permeabilizing one or more nuclei; b. contacting the one or more nuclei with(ii) a first transposase; wherein the first transposase is loaded with a nucleic acid comprising a first tag, wherein the first tag comprises a first restriction site and a barcode selected from a first set of barcodes; c. initiating a tagmentation reaction, resulting in the generation of genomic DNA fragments comprising the first tag; (e.g. Cells are fixed and nuclei are isolated for In situ tagmentation and reverse transcription. Barcoded Tn5 then added to initiate tagmentation reaction, generating genomic DNA fragments that comprise the barcode [Method and Fig. 1a])
d. reverse transcribing the RNA in the one or more nuclei using primers comprising a second tag, wherein the second tag comprising a second restriction site and the barcode of the first tag, resulting in the generation of cDNA comprising the second tag; (reverse transcription was performed using barcoded RT primers and Reverse Transcriptase to generate barcoded cDNA from mRNA. Additionally, restriction sites are incorporated into the adapters during the combinatorial barcoding process [Method and Fig. 1a])
e. contacting the one or more nuclei with a ligase and a third tag comprising a second barcode selected from a second set of barcodes, resulting in the generation of genomic DNA fragments comprising a first tag and a third tag and cDNA comprising a second tag and a third tag; (Nuclei were transferred to Ligation Mix for the third barcode to be ligated to both DNA fragments and cDNA [Method and Fig. 1a])
f. lysing the one or more nuclei; g. fusing a polynucleotide tail to the DNA and cDNA, generating polynucleotide tailed DNA and cDNA; (e.g. The nuclei are lysed, then TdT enzyme is added to the mixture to add a homopolymer tail to both genomic DNA (gDNA) and cDNA. [Method and Fig. 1a])
h. amplifying the polynucleotide tailed DNA and cDNA; i. dividing the amplified polynucleotide tailed DNA and cDNA into a DNA library and an RNA library; (e.g. pre-amplification mix is used to amplify barcoded DNA/cDNA libraries. The mixture then purified after amplification and split into two tubes for DNA and RNA library [Method and Fig. 1a])
j. for the DNA library: ii. contacting the DNA end with a sequencing adaptor and a ligase, resulting in the generation of amplified polynucleotide tailed DNA comprising the sequencing adaptor; iii. cleaving the amplified polynucleotide tailed cDNA with an enzyme recognizing the second restriction site; (e.g. added 1.5 µl SbfI-HF (NEB, R3642) per 100 ng amplified product to the DNA, then ligate additional sequencing adaptor handle to both DNA and RNA libraries[Method and Fig. 1a])
k. for the RNA library: i. cleaving the amplified polynucleotide tailed DNA with a restriction enzyme recognizing the first restriction site; ii. contacting the amplified polynucleotide tailed cDNA with a second transposase loaded with a nucleic acid comprising a sequencing adaptor and initiating a tagmentation reaction, resulting in the generation of amplified polynucleotide tailed cDNA comprising the sequencing adaptor; (e.g. added 0.75 µl NotI-HF (R3189) per 100 ng amplified product to the RNA tube, then ligate additional sequencing adaptor handle to both DNA and RNA libraries[Method and Fig. 1a])
l. sequencing the molecules in the RNA library and the DNA library;m. correlating the RNA library and the DNA library for each of the one or more nuclei. [Fig. 1a-g]
However, Zhu does not disclose: a(i) an antibody that binds to a chromatin-associated protein or chromatin modification; h. the primers used for the amplification of the DNA comprises a third restriction site and wherein the third restriction site is recognized by an endonuclease; and j (i) cleaving the amplified polynucleotide tailed DNA with a restriction an endonuclease recognizing the third restriction site;
Kaya-Okur discloses a method for profiling chromatin components, wherein chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. The transposase efficiently tagments and generates sequencing-ready fragment libraries [abstract and Fig. 1]
Lundin discloses type IIS restriction enzymes (endonuclease class that cleaves at the third restriction site) have been extensively utilized for nucleic acid analysis, e.g. for gene expression analysis and massively parallel sequencing due to their shifted cleavage property, enables manipulation of unknown nucleotide compositions enabling analysis of novel sequences [Abstract].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to include the antibody-directed chromatin-protein targeting of Kaya-Okur and the third restriction site that utilize type IIS restriction enzymes cleavage strategy because Zhu already seeks to analyze gene-regulatory information from single cells using barcoded tagmentation and sequencing, while Kaya-Okur provide a targeted chromatin profiling method that target specific chromatin associate proteins or modifications. The addition of Kaya-Okur’s antibody directed transposase targeting would have predictably allowed Zhu’s method to obtain beyond general chromatin accessibility information, more targeted chromatin associated protein or specific chromatin modifications from the same type of nuclei/cell. Additionally, a killed artisan would have been motivated to incorporate type IIS restriction site into Zhu’s adapter because as disclosed by Ludin, this class of endonuclease cleave outside their asymmetric recognition sites, enables manipulation of unknown nucleotide compositions enabling analysis of novel sequences at high-efficiency [Lundin introduction]. The proposed combination represents using known molecular techniques for their established purposes in a compatible sequencing library workflow. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143)
Regarding claim 2 is rejected for substantially the same reason set forth in claim 1. Claim 2 recites the same overall method steps as claim 1, except the third restriction site is introduced during amplification of cDNA rather than gDNA, and the downstream RNA-library and DNA-library steps are correspondingly rearranged. These differences do not introduce new limitation and overcome the rejection discussed above because Zhu, Kaya-Okur, and Lundin disclose the same barcoded single cell DNA/cDNA library workflow, antibody directed transposase targeting, and type IIS restriction-site cleavage strategy.
Regarding claims 5 and 6, Zhu further discloses ligating a fourth tag comprising a third barcode after step e, and step recited in claim 5 repeated one or more times. (e.g. Fig. 1a shows multiple rounds of multiplex barcode ligation after permeabilizing, tagmentation, reverse transcription, and second barcode ligation)
Regarding claims 11 and 12, Lundin further discloses the third restriction site is recognized by a type IIS endonuclease, selected from the group consisting of FokI, AcuI, AsuHPI, BbvI, BpmI, BpuEI, BseMII, BseRI, BseXI, BsgI, BslFI, BsmFI, BsPCNI, BstV1I, BtgZI, EciI, Eco57I, FaqI, GsuI, HphI, MmeI, NmeAIII, SchI, TaqII, TspDTI, and TspGWI. [Abstract]
Regarding claims 13, Zhu further discloses the polynucleotide tail is fused to the DNA and cDNA by contacting the DNA and cDNA with (i) a terminal deoxynucleotidyltransferase (TdT) [Method].
Regarding claims 14-16 are rejected for the same reason as claim 13 because claim 13 recites alternative tailing methods, and claim 14-16 further limit certain alternatives without excluding the TdT option. Thus, Zhu disclosure of TdT-mediate tailing satisfies the method encompasses by claim 13 and applies to claims 14-16.
Regarding claim 18, Kaya-Okur further discloses the chromatin-associated protein is a transcription factor protein is a histone protein [abstract].
Regarding claim 19, Kaya-Okur further discloses the chromatin modification is a histone modification [abstract]
Regarding claim 20, Zhu further discloses the nuclei are obtained from a mammal.(e.g. HEK293T [method])
Regarding claim 21 and 24, Zhu further discloses a multi-round of split and pool barcoding method where nuclei are divided, pooled, barcoded, divided again, pooled again, barcoded again for many rounds. Finally lysed, allowing DNA and cDNA from each nucleus to have combinatorial barcode [Main section and Fig. 1a]
Regarding claims 23, Zhu further discloses the pooling, the dividing and the contacting the sub-samples with a ligase and a tag comprising an additional barcode are repeated one or more times[Fig. 1]
Zhu et al., Kaya-Okur et al., Lundin et al., and Fernandez et al.
Claim(s) 4 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. (Nat Struct Mol Biol 26, 1063–1070 (2019), disclosed in IDS )in view of Kaya-Okur et al. (Nat Commun 10, 1930 (2019), disclosed in IDS), Lundin et al. (PLoS One. 2015;10(1):e0117059), and Fernandez et al. (US9938524B2, published April 23rd 2015)
Regarding claim 4 and 22, Kaya-Okur further discloses in step (b) of the method: a. the one or more nuclei are first contacted with the antibody and then contacted the first transposase, wherein the first transposase is linked to a binding moiety that binds to the antibody; (e.g. 1) nuclei first contact with the antibody, 2) antibody then binds to chromatin, and 3) finally, the nuclei are contacted with transposase. [Fig. 1]). However, Kaya-Okur does not disclose the alternative ways of introducing antibody-transpose into nuclei as recited in (b) and (c).
Fernandez teaches the antibody is first incubated with the first transposase linked to a binding moiety that binds to the antibody; and the one or more nuclei are contacted with the antibody bound to the transposase; discloses the one or more nuclei are contacted with an antibody that is covalently linked to the first transposase. (Under BRI, “linked” can be reasonably interpret as covalent linkage. Thus, Fernandez teaches an antibody-transposase covalent conjugate of both (b) and (c) [claim 1 of US 9938524 B2 patent])
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to substitute Kaya-Okur’s way of introducing antibody- transpose system with Fernandez because both references use antibody directed transposase targeting to generate chromatin-associated sequencing libraries. Kaya-Okur teaches sequential antibody binding followed by transposase recruitment, while Fernandez teaches pre-associated antibody and transposase conjugates. A skilled artisan would have been motivated to use Fernandez’s conjugate format as an alternative way to tether transposase to antibody with the predictable result of directing transposase activity to the selected chromatin associated protein or modification.
Allowable Subject Matter
Claim 17 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent from including all of the limitations of the base claim and any intervening claims.
The closest prior art reference to claim 17 is Moks (Eur. J. Biochem., 156: 637-643 (1986)). Moks teaches IgG-binding properties of the N-terminal portion of staphylococcal protein A, but provide no teaching or suggestion that such activity should be utilized in parallel analysis of individual cells for profiling of transcriptome and chromatin.
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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