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 and Action Summary
This action is in response to the papers filed on November 21, 2025.
Claims 1-6, 9-10, 12, 15-19, 22, 23, 25, and 28-30 are pending in this application.
Claims 8 and 21 were canceled and claims 29-30 were added in the response filed on November 21, 2025.
Claims 1-6, 9-10, 12, 15-19, 22, 23, 25, and 28-30 are under examination. No other claims are currently pending in the present application.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections of record to the specification are withdrawn in view of the amendments to the specification.
The 102(a)(1) rejections of record over Rudi et al., Jacobsen et al., and Bailey et al. are withdrawn in view of the amendments to the claims, particularly the new requirement that the nucleic assay recited in step (c) of independent claims 1 and 16 “is a whole genome amplification or whole transcriptome amplification”.
The 103 rejections of record over Bailey et al. in view of Ehrich et al. have been withdrawn in view of the amendments to the claims, particularly the new requirement that the nucleic assay recited in step (c) of independent claims 1 and 16 “is a whole genome amplification or whole transcriptome amplification”.
The 112(b) indefiniteness rejections of record are withdrawn in view of the amendments to the claims.
Priority
The present application, filed on February 23, 2023 is a 371 of PCT/US2021/052337, filed on September 28, 2021, which claims the benefit of U.S. Provisional Application 63/089,719, filed on October 9, 2020. Therefore, the effective filing date of the present application is determined to be October 9, 2020.
Drawings
The drawings filed on February 23, 2023 are acceptable.
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.
Claims 9-10 and 22-23 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This is a new grounds of rejection necessitated by the amendments to the claims.
Claims 9 and 22 recite “the method further comprises performing the nucleic acid assay for determining at least one genetic variant”. Claims 1 and 16 recite “reagents for performing a nucleic acid assay… wherein the nucleic assay is a whole genome amplification or whole transcriptome amplification”, however these independent claims, upon which claims 9 and 22 respectively depend, do not recite “a nucleic acid assay for determining at least one genetic variant”. Additionally, there is no “determining at least one genetic variant” step inherent to “whole genome amplification or whole transcriptome amplification”. Therefore, there is insufficient antecedent basis for this limitation in the claims. Furthermore, it is unclear whether “the nucleic acid assay for determining at least one genetic variant” is simply intended to refer to “whole genome amplification or whole transcriptome amplification” and is merely an intended use of “whole genome amplification or whole transcriptome amplification” OR if this term is intended to require performing an additional, unspecified nucleic acid assay that accomplishes “determining at least one genetic variant” such as: allele-specific PCR, sequencing and alignment to a reference genome or control, hybridization of allele-specific probes to the amplified nucleic acids, other such assays known in the art for determining at least one genetic variant, or something else entirely.
Claims 10 and 23 recite the limitation “the at least one genetic variant is selected from the group comprising aneuploidy, mosaicism, single nucleotide polymorphism, and any combination thereof”. This limitation is indefinite for the following reasons: a) A Markush grouping is a closed list of alternatives wherein the selection is made from a group “consisting of” rather than “comprising” or “including” the alternative members. Abbott Labs., 334 F.3d at 1280, 67 USPQ2d at 1196. Given the extremely broad nature of the genus “genetic variant” and/or the alternative “and any combination thereof”, and the use of the open-ended word “comprising”, it is unclear what other alternatives are intended to be encompassed by the claim. (See MPEP 2173.05(h)).
Applicant is reminded that NO NEW MATTER can be added to the disclosure.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-6, 9-10, 12, 15-18, 22, 23, 25, and 28-30 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Macaulay et al., “G&T-seq: parallel sequencing of single-cell genomes and transcriptomes” Nature Methods Vol. 12 No. 6 pages 519-525 (published April 27, 2015).
This is a new grounds of rejection necessitated by the amendments to the claims.
Regarding claim 1, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules using a plurality of beads for binding the nucleic acid molecules, and adding reagents for whole-transcriptome amplification to the bound nucleic acid molecules (“on-bead” Whole-transcriptome amplification) (Macaulay et al., figure 1a, reproduced below for convenience).
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Therefore, Macaulay et al. teach all of the positively recited method steps of claim 1.
Regarding claim 2, Macaulay et al. teach “Genomic DNA present in the pooled supernatant and wash buffer from the mRNA-isolation step (see figure 1a above) was precipitated on Ampure Beads… and eluted directly into the reaction mixtures for amplification by either MDA… or PicoPlex (i.e. whole genome amplification) (Macaulay et al., page 523, column 2, paragraph 5).
Regarding claim 3, Macaulay et al. teach manually selecting or flow sorting single cells into lysis buffer (i.e. lysing the cells) and processed immediately by adding the magnetic Biotin-oligo-dT beads (see figure 1a) directly to the cell lysate, binding mRNA to the beads, collecting non-bound DNA, washing the beads, collecting the wash buffer, and resuspending the beads in reagents for whole transcriptome amplification (Macaulay et al., page 523, column 1-column 2 bridging paragraph – column 2, paragraph 4) (i.e. steps a-c are performed in a single vessel).
Regarding claim 4, Macaulay et al. teach on-bead whole transcriptome amplification (i.e. binding the nucleic acids and adding reagents for performing… whole transcriptome amplification simultaneously) (Macaulay et al., page 523, column 1-column 2 bridging paragraph – column 2, paragraph 4 and figure 1a).
Regarding claim 5, Macaulay et al. teach the cells are lysed in RLT Plus buffer (Qiagen) (i.e. a lysis reagent) (Macaulay et al., page 523, column 1, paragraph 5).
Regarding claim 6, Macaulay et al. do not teach using a lysis reagent comprising a protease. However, it is noted that the lysis step recited by claim 1 is an optional step and is therefore not required by the claims. Furthermore, claim 6 does not require that this step occurs, but merely limits the composition of the optional lysis buffer not required by claim 1.
Regarding claims 9-10, Macaulay et al. teach calling single nucleotide variants (i.e. single nucleotide polymorphisms) from the single-cell RNA-seq data generated using the method described in the rejection of claim 1 above (Macaulay et al., figure 1a and page 525, column 2, paragraph 2).
Regarding claim 12, Macaulay et al. teach that the beads are magnetic beads (Macaulay et al., figure 1a).
Regarding claim 15, Macaulay et al. teach the following samples: cultured breast cancer cells, cultured B lymphoblastoid cells (both derived from the same patient) (Macaulay et al., page 519, column 2, paragraph 2), cultured mature cortical neurons differentiated from induced pluripotent stem cells (i.e. somatic cells) (Macaulay et al., page 523, column 1, paragraph 4), and single blastomere cells of eight-cell cleavage-stage mouse embryos (i.e. germ cells, fetal cells, somatic cells) (Macaulay et al., page 520, column 2, paragraph 2).
Regarding claim 29, which depends from claim 1, Macaulay et al. teach performing whole transcriptome amplification (i.e. the nucleic acid assay) (Macaulay et al., figure 1a).
Regarding claim 16, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules (i.e. DNA) using a plurality of beads for binding the nucleic acid molecules (i.e. Ampure beads), and eluting the bound nucleic acids (DNA) directly into reaction mixtures for whole genome amplification (i.e. MDA or PicoPlex) (Macaulay et al., figure 1a, and page 523, column 2, paragraph 5).
Regarding claim 17, Macaulay et al. teach performing PCR immediately after the mRNA capture and on-bead cDNA synthesis steps by adding PCR mastermix to the reverse-transcription reaction mixture and thermal cycling comprising 98 degree Celsius denaturation steps (i.e. eluting the nucleic acids into a reagent for a nucleic acid assay while the beads remain present in the reaction mixture) (Macaulay et al., page 523, paragraphs 2-4).
Regarding claim 18, Macaulay et al. teach isolating the nucleic acids by binding the nucleic acids to Ampure beads and eluting directly into reaction mixtures for whole genome amplification (i.e. steps b and c are performed simultaneously) (Macaulay et al., page 523, column 2, paragraph 5).
Regarding claims 22-23, Macaulay et al. teach determining genetic variants including trisomy (i.e. aneuploidy, single nucleotide variants, and mosaicism (i.e. a subset of cells having an aneuploidy not common to the entire population of cells)) (Macaulay et al., page 520, column 1-2 bridging paragraph; page 522, column 1, paragraph 2).
Regarding claim 25, Macaulay et al. teach that the beads are Ampure beads (i.e. magnetic beads).
Regarding claim 28, Macaulay et al. teach the following samples: cultured breast cancer cells, cultured B lymphoblastoid cells (both derived from the same patient) (Macaulay et al., page 519, column 2, paragraph 2), cultured mature cortical neurons differentiated from induced pluripotent stem cells (i.e. somatic cells) (Macaulay et al., page 523, column 1, paragraph 4), and single blastomere cells of eight-cell cleavage-stage mouse embryos (i.e. germ cells, fetal cells, somatic cells) (Macaulay et al., page 520, column 2, paragraph 2).
Regarding claim 30, Macaulay et al. teach performing whole genome amplification; MDA and PicoPlex (i.e. the nucleic acid assay) (Macaulay et al., figure 1a).
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.
Claims 1-6, 9-10, 12, 15-19, 22, 23, 25, and 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Macaulay et al., “G&T-seq: parallel sequencing of single-cell genomes and transcriptomes” Nature Methods Vol. 12 No. 6 pages 519-525 (published April 27, 2015) in view of Bailey et al., “Single-Tube Analysis of DNA Methylation with Silica Superparamagnetic Beads”, Clinical Chemistry 56:6 pp. 1022-1025 (2010).
This is a new grounds of rejection necessitated by the amendments to the claims.
The teachings of Macaulay et al. regarding claims 1-6, 9-10, 12, 15-18, 22, 23, 25, and 28-30 are detailed in the 102(a)(1) rejection above.
Regarding claim 1, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules using a plurality of beads for binding the nucleic acid molecules, and adding reagents for whole-transcriptome amplification to the bound nucleic acid molecules (“on-bead” Whole-transcriptome amplification) (Macaulay et al., figure 1a, reproduced below for convenience).
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Regarding claim 16, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules (i.e. DNA) using a plurality of beads for binding the nucleic acid molecules (i.e. Ampure beads), and eluting the bound nucleic acids (DNA) directly into reaction mixtures for whole genome amplification (i.e. MDA or PicoPlex) (Macaulay et al., figure 1a, and page 523, column 2, paragraph 5).
Therefore, Macaulay et al. teach all of the positively recited method steps of independent claims 1 and 16.
Regarding claims 6 and 19, Macaulay et al. do not teach that “the lysis reagent comprises a protease” or “the lysis… is performed using a protease”. Rather, Macaulay et al. lyse cells in RLT Plus buffer (Qiagen) (a lysis buffer comprising Guanidine HCl) (Macaulay et al., page 523, column 1, paragraph 5).
However, Bailey et al. teach methods comprising on-bead determination of DNA methylation (i.e. preparing nucleic acid molecules from a sample) comprising lysing the cells with a lysis reagent comprising Guanidine HCl and Protease K (i.e. a protease).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have added a protease, such as Protease K, to the lysis buffer taught by Macaulay et al. The ordinary artisan would therefore have recognized that lysis reagents comprising Guanidine HCl or Guanidine HCl are recognized equivalents known for the same purpose (i.e. breaking open, “lysing” cells). The ordinary artisan would have had a reasonable expectation that lysing cells with a lysis reagent comprising Protease K would have successfully released nucleic acids.
Claims 1-6, 9-10, 12, 15-19, 22, 23, 25, and 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Macaulay et al., “G&T-seq: parallel sequencing of single-cell genomes and transcriptomes” Nature Methods Vol. 12 No. 6 pages 519-525 (published April 27, 2015) in view of Hou et al., “Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas” Cell Research (2016) 26:304-319.
This is a new grounds of rejection necessitated by the amendments to the claims.
The teachings of Macaulay et al. regarding claims 1-6, 9-10, 12, 15-18, 22, 23, 25, and 28-30 are detailed in the 102(a)(1) rejection above.
Regarding claim 1, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules using a plurality of beads for binding the nucleic acid molecules, and adding reagents for whole-transcriptome amplification to the bound nucleic acid molecules (“on-bead” Whole-transcriptome amplification) (Macaulay et al., figure 1a, reproduced below for convenience).
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Regarding claim 16, Macaulay et al. teach methods of preparing nucleic acid molecules from a sample (i.e. single cells) comprising: lysing the cells (i.e. the sample), isolating the released nucleic acid molecules (i.e. DNA) using a plurality of beads for binding the nucleic acid molecules (i.e. Ampure beads), and eluting the bound nucleic acids (DNA) directly into reaction mixtures for whole genome amplification (i.e. MDA or PicoPlex) (Macaulay et al., figure 1a, and page 523, column 2, paragraph 5).
Therefore, Macaulay et al. teach all of the positively recited method steps of independent claims 1 and 16.
Regarding claims 6 and 19, Macaulay et al. do not teach that “the lysis reagent comprises a protease” or “the lysis… is performed using a protease”. Rather, Macaulay et al. lyse cells in RLT Plus buffer (Qiagen) (a lysis buffer comprising Guanidine HCl) (Macaulay et al., page 523, column 1, paragraph 5).
However, Hou et al. teach methods comprising lysing single cells for preparing nucleic acid molecules for nucleic acid assays comprising whole genome amplification (Hou et al., page 314, column 1, paragraph 3) wherein the single cells are lysed in “a mild lysis protocol” comprising treating the cells with a “soft buffer” comprising protease (Hou et al., page 305, column 1, paragraph 3 and page 315, column 1-2 bridging paragraph).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have substituted the RLT lysis buffer taught by Macaulay et al. for the “soft buffer” comprising protease in a “mild lysis protocol” taught by Hou et al. The ordinary artisan would have been motivated to substitute the mild lysis conditions taught by Hou et al. into the methods taught by Macaulay et al. because Hou et al. explicitly teaches that the mild lysis conditions are compatible with conventional single-cell methods (Hou et al., page 314, column 1, paragraph 3) including whole genome amplification, while allowing for simultaneous measurement of genome, methylome, and transcriptome information in single cells (Hou et al., page 314, column 1, paragraph 2).
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, 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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682