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 Claims
Applicant' s amendment filed 05/22/2026 is acknowledged. Claims 16, 18-21, 23, and 27-33 have been amended. Claims 36-37 have been added. Claims 1-15 and 17 are cancelled. Claims 16 and 18-37 are pending in the instant application and the subject of this final office action.
All of the amendments and arguments have been reviewed and considered. Any rejections or objections not reiterated herein have been withdrawn in light of amendments to the claims or as discussed in this office action.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Action
Status of Prior Rejections/Objections:
The objection to the specification is withdrawn in view of the amendment to the specification.
The 112(b) rejections directed to claims 16-18, 21, 23, and 28 are withdrawn in view of the amendments to or cancellation of the claims.
The prior art rejection(s) under 35 USC 102 directed to claim(s) 16, 18-20, 22-23, 26-29, and 34 as being anticipated by Gawad are withdrawn in view of the amendments to the claims.
Each of the prior art rejection(s) under 35 USC 103 is withdrawn in view of the amendments to the claims. See new art rejections in view of the amendments.
Each of the double patenting rejections directed to copending applications 17/631,130; 18/860,539; 18/861,541 and 18/882,493 and US Patent 11,905,553 have been modified as necessitated by Applicant’s amendments.
Priority
The instant application claims priority to provisional applications 63/338,669 and 63/406,862. These applications lack support for the limitations of claims 29-33, which have been given a priority date of 05/04/2023 based on the PCT/US2023/021073. The other claims 16, 18-28, and 34-37 have been given a priority date of 05/05/2022.
New Ground(s) of Rejections
The new ground(s) of rejections were necessitated by applicant’s amendment of the claims.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claim 30, the term “a 1x coverage” in the phrase “a 1x coverage of at least 0.95" was interpreted to include “the percentage of the reference genome that is covered by at least one read”, supported by para [0026] and Table 9. For applications directed to less than a whole genome (e.g., targeted sequencing), as such is not required by the claim, it is further interpreted to encompass at least “the percentage of the reference sequence targeted that is covered by at least one read”.
Claim Rejections - 35 USC § 103
Claim(s) 16, 18-20, 22-23, 26-30, 32, and 34-37 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025) in view of Saxonov (WO 2012/149042 A2).
Regarding claims 16, 27, and 34, Gawad teaches a method comprising:
contacting a sample comprising a single cell with a lysis buffer/mix comprising exonuclease-resistant random primers (para [00154-155]; para [00184]);
adding to the cell lysate/lysis buffer a quenching buffer and an amplification mix comprising dNTPs and polymerase (para [00154-155]; instant claim 34), including alpha-thio-ddNTPs (para [00155]; instant claim 27), and amplifying [genomic] DNA from the single cell (para [00155]; see also para [00160] which shows alignment to hg19, i.e., genomic DNA). See also para [00185].
sequencing the amplification products (para [00158-00164]).
Gawad teaches that its buffers may comprise salts including magnesium chloride (para [00123]; see para [00154]) and recites that phi29 DNA polymerase is believed to have a lower error rate (para [00182] and [00107]) and that genetically modified phi29 is suitable (para [00107]).
Regarding claim 18, Gawad teaches amplifying for 8 hours before terminating the reaction (para [00154] and [00155]).
Regarding claim 19, Gawad teaches following amplification with addition of end repair and A-tailing (ERAT) mixture (Fig. 2A and 6; para [00158])
Regarding claim 20, Gawad teaches no purification steps amongst steps (a) and (b) (para [00154-155], [00185]).
Regarding claim 22-23, Gawad teaches the lysis is alkaline lysis (Fig. 1B-E) [i.e., that the lysis buffer comprises a base]. Gawad teaches that sodium hydroxide [i.e., a base] may be used (para [00122]; see also, e.g., para [0084] re: targets)).
It is noted that claim 23 limits the identity of the buffering agent, but does require that the lysis buffer comprise a buffering agent.
Regarding claim 26, Gawad teaches analyzing at least 100 cells (para [0097]; see also [00205]).
It is noted that the claim encompasses bulk or the number of single cells under BRI.
Regarding claim 28, Gawad teaches that the amplification mix contained alpha-thio-ddNTPs at equal ratios at a concentration of 1200 uM (para [00155]), interpreted to be ddATP, ddCTP, ddGTP, and ddTTP, each at a concentration of 400 uM [i.e., 0.4 mM].
Gawad teaches optimizing alpha-thio-ddNTP concentrations for droplet volumes (para [00185]; see also pg. 57, line 2).
Regarding claims 29-30 and 32, Gawad teaches achieving an allele balance of 1.0 (Fig. 4B, panel 4, wherein the minimum and maximum are, respectively, greater than 0 and less than 1, i.e., both alleles are detected in all regions of known heterozygosity in the reference sequence; see instant Table 9, instant para [00114], and para [00128]; instant claim 29). Gawad teaches that PTA has more even amplification across the genome but across two alleles in the same cell (para [00180]).
Gawad teaches a 1x genomic coverage in cells analyzed by the method of 97% (Table 2; see also para [00204-205] and Fig. 11; instant claim 30). Gawad teaches that [single cell] PTA had the highest percentage of reads aligned to the genome as well as the highest mapping quality, using the same variant calling pipeline (para [00177]). Gawad teaches that PTA approaches the two bulk samples at every depth and that PTA had uniform coverage (para [00178]).
Gawad teaches an SNV sensitivity of about 0.85 (Fig. 5E; instant claim 32).
Gawad teaches that base change patterns appear polymerase-dependent and that Gawad’s PTA further suppresses error propagation wherein the error-rate for Gawad is based on phi29 (para [00182]; Fig. 5H and 5I).
Gawad teaches scaling the method for massively parallel DNA sequencing (para [00184]) and transitioning the amplification into droplets, wherein the concentrations of reagents for the low nanoliter volumes of droplets is optimized (para [00185]).
Gawad fails to explicitly teach:
that the magnesium is present at a concentration of 2-6 mM (claim 16) or 2-4 mM (claim 36) and therefore the corresponding dNTP to magnesium ratio (claim 37);
that each of the one or more terminator nucleotides is each present at a concentration of less than 0.3 mM (claim 28).
Saxonov teaches methods for nucleic acid analysis (title), including single cell genomic sequencing comprising MDA (para [00176-00179]), wherein said amplification may use phi29 and occur within partitions/droplets for performing an amplification reaction (para [00134]). Saxonov teaches that such multiplexing is useful for evening out biological variation (para [00236]).
Saxonov teaches that said droplets may comprise an aqueous phase of an emulsion comprising a buffered solution and reagents for performing an amplification reaction (para [0087]), including MgCl2 of about or less than about 1.0, 2.0, 3.0, or 5.0 mM (para [0091]; instant claims 16 and 36).
Saxonov also teaches adding non-canonical nucleotides to the partitions/droplets at a concentration of less than about 300 uM [i.e., 0.3 mM] and teaches various ratios relative to canonical dNTPs (para [0090] and [00121]; instant claim 28).
Saxonov teaches that the nucleotides including dATP, dCTP, dGTP, and dTTP, are in concentrations of about 100-700 uM each [i.e. 0.1-0.7 mM]; instant claim 35).
Given the concentrations of MgCl2 and the dNTPs, Saxonov at least a ratio of dNTPs to magnesium of about 1.4 (e.g., (0.7 + 0.7 + 0.7 + 0.7) /2.0; instant claim 37).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the method of Gawad for droplets and in doing so, have utilized at least the known concentration(s) of magnesium, dNTPs, and ratio(s) thereof of Saxonov, as Saxonov is a “comparable” method improved in the same way. The artisan would have been so motivated at least because transition to droplets is taught by Gawad and Saxonov teaches such multiplexing is useful for evening out biological variation (instant claims 16 and 35-37).
Further, such is determined to be a matter of routine optimization of such buffer(s), as taught/suggested by Gawad and/or Saxonov as at least optimizing the buffer concentrations and wide ranges of values are taught. See MPEP 2144.05(II).
It further would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have similarly optimized the terminator nucleotides, as the amount of Gawad is interpreted to be close to that value claimed and Saxonov presents a similar strategy of non-canonical bases and teaches the claimed range and teaches optimizing relative to the canonical bases (instant claim 28). See MPEP 2144.05(II).
The artisan could have performed such an optimization as Gawad teaches optimizing and characteristics for success of the assay and both are directed to single cell DNA sequencing that may be performed in droplets based on the same MDA that may be performed with the same polymerase.
It is noted that where claims are directed to sequencing results, while the sequencing results of Gawad are not explicitly taught to be those with a particular concentration of magnesium, such results would be obvious to optimize for in view of Gawad and be expected to be achievable at least because Gawad teaches that base change patters are polymerase-dependent and that the reduced error propagation is based on the method itself (e.g., the inclusion of the terminator) (instant claims 29-30 and 32).
Claim(s) 21-25 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025) in view of Saxonov (WO 2012/149042 A2) as applied to claims 16, 18-20, 22-23, 26-30, 32, and 34-37 above, and in view of Dean (US 7,074,600 B2; published 07/11/2006).
Regarding claims 21, in the method of Gawad in view of Saxonov, Gawad teaches controlling the number of amplicons produced by the amount of time the amplification is allowed the proceed (para [00126]). Gawad teaches that errors may be propagated from daughter amplicons during subsequent amplifications (para [00106]).
Dean teaches that by using a sufficient number of primers, only a few rounds of replication are required to produce hundreds of thousands of copies of the nucleic acid sequences of interest, wherein target amplicons can be produced in 10 minutes or 60 minutes, based on the amount of desired “rounds” of amplification (col 47, para 1).
Therefore, it would have been obvious to conduct amplification for less than 60 min or the addition of lysis buffer and amplification for a combined total of 60 minutes if the artisan desired only the number of amplicons produced during that time period and/or to reduce the potential for propagation of errors from parent amplicons, as taught by Gawad and Dean, as such is subject to routine optimization and omission of further “cycles” if not desired. See MPEP 2144.04 (II)(A) and 2144.05(II).
Regarding claim 22-25, in the method of Gawad in view of Saxonov, Gawad teaches the lysis is alkaline lysis (Fig. 1B-E), and that the buffers may contain Tris-HCl, EDTA (para [00123]).
Gawad teaches optimization of reagent concentrations (para [00185]).
Saxonov teaches a pH of the partition may be 11-12.5 (para [0088]) and that buffering agents of the partition may include HEPES, BES, TEA, MOPS, TES, MOBS, TAPSO, Trizma [triz(hydroxymethyl)aminomethane], HEPPSO, POPSO, or TEA (para [0088)].
However, Gawad and Saxonov fails to teach specific concentrations of buffer components and/or that these are components of a lysis buffer.
Dean rectifies this by teaching a method using alkaline lysis buffer for MDA (entire document, e.g., col 2, para 1-2) comprising adding a lysis solution (col 18, para 2), wherein the lysis solution has a pH of about 11.0 to 13.0 (col 18, para 3; instant claim 25) and component concentrations range from 10 mM to about 500 mM (col 19, line 5; instant claim 24), including 400 mM KOH, 100 mM DTT, 10 mM EDTA (claim 8; instant claims 22 and 24) and at least one buffering agent, wherein the one or more buffering agent may be HEPES, MOPS, TES, or Tris (claims 14 and 15; instant claims 22-23). It is noted for the sake of compact prosecution that Dean recites that KOH and NaOH are substitutes (claim 5).
Dean further teaches mixing with a stabilization/denaturing [neutralization] solution (entire document, e.g., col 38, para 1-3; D. Stabilization Solution; E. Denaturing Solution) and mixing with polymerase and nucleotides (entire document, e.g., O. Mixtures, Method; claims 1-202).
Dean teaches that the amount of lysis solution and pH of the solution should be optimized together (col 18, para 2).
Dean teaches that the methods allow amplification of target nucleic acids from whole genomes and highly complex nucleic acid samples (col 8, Detailed Description, para 1), that the genome can be amplified through simple cell lysis techniques and amplification performed on crude lysates (col 6, para 1), and that the method generates plentiful amounts of amplified DNA that faithfully reproduces the locus representation frequencies of the starting material (col 42, para 5).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined method of Gawad in view of Saxonov with the buffer components and component concentrations utilized in Dean, motivated by the desire to faithfully reproduce allele [locus] frequencies of the starting material and generate plentiful amounts of amplified DNA, as taught by Dean. There would have been a strong expectation for success as each is compatible with alkaline and MDA-based methods.
Further, such buffer components concentrations are identified as routinely optimized variables, for the reasons discussed in Dean and/or Gawad and as noted above. See MPEP 2144.05(II).
Claim(s) 31 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025) in view of Saxonov (WO 2012/149042 A2) as applied to claims 16, 18-20, 22-23, 26-30, 32, and 34-37 above, and further in view of Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf).
Regarding claims 31, in the method of Gawad in view of Saxonov, Gawad teaches that primers of the invention may be targeted to a specific genomic region (para [00116]). Gawad teaches using a panel targeting specific genes (para [00209]).
Gawad also teaches variant calling using a pipeline (para [00177]) and determining SNV sensitivity (para [00179-180]) and SNV specificity (para [00181-182]).
Gawad fails to teach a precision [sample variant concordance; see instant Table 9] of at least 0.99.
Takara rectifies this by teaching a method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2); Takara teaches that the workflow is economical (pg. 1, para 3). Takara teaches applying the method to a cell line and obtaining 100% variant concordance [i.e., sensitivity] (pg. 1, para 4). Takara teaches that the workflow may be applied to one cell (pg. 1, Workflow).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the method of Gawad in view of Saxonov with the targeted panel method of Takara, motivated by the desire to reduce time, labor, and costs, as taught by Takara, as well as to improve the pr. There would have been a strong expectation for success as Gawad also suggest applying a target-specific panel and both are directed to variant calling in low-input samples.
Alternatively, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the variant calling pipeline of Gawad and the optimization strategies of Gawad and Saxonov to have optimized the method to calculate and achieve such a precision, motivated by the desire to achieve a competitive product. Such a result would have been predictable given the high true positive and low false positive values indicated by the taught sensitivity and specificity of the method of Gawad, comparative performance versus other amplification strategies taught by Gawad, and the means indicated for further optimization in Gawad and Saxonov.
Claim(s) 33 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025) in view of Saxonov (WO 2012/149042 A2) as applied to claims 16, 18-20, 22-23, 26-30, 32, and 34-37 above, and further in view of Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.).
Regarding claims 33, in the method of Gawad in view of Saxonov, Gawad teaches a base sensitivity of about 0.85 of the bulk variants called (Fig. 5E). Gawad teaches that the method may be applied to a combined method of gDNA and mRNA analysis (entire document, e.g., Example 4). Gawad teaches base change patterns may be polymerase-dependent (para [00182]; [0038]) and choosing among multiple polymerases such as a genetically modified phi29 (para [00107]). Gawad teaches that primers of the invention may be targeted to a specific genomic region (para [00116-117]) and that the amplicon libraries may be further amplified, including by PCR (para [00113]). Gawad teaches using a panel targeting specific genes (para [00209]).
Gawad and Saxonov fail to teach an SNV sensitivity of 0.99.
Rodriguez-Meira teaches a method of single cell sequencing that dramatically increases the sensitivity of mutation detection by modifying template-switching protocols by adding target-specific primers for cDNA and gDNA to the RT and DNA amplification steps and utilizing modified enzymes (pg. 1293, Results, para 1). Rodriguez-Meira teaches that combining mutation detection of both mRNA and gDNA targeting allowed for detection of all SNVs analyzed in 98.4% of cells (pg. 1295, col 1, para 2).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined method of Gawad with the targeted amplification of SNV targets and combination with mRNA read analysis of Rodriguez-Meira, motivated by the desire to dramatically increase the sensitivity of mutation detection. There would be a strong expectation of success as both are directed to single cell methods that may comprise mRNA and gDNA analysis and targeted amplification.
Further, as Gawad teaches multiple polymerases including an optimized version phi29 and both Gawad and Rodriguez-Meira teaches optimizing polymerases to reduce errors/improve detection, it would further be obvious to optimize the combined method further by routinely optimizing the choice of polymerase, wherein it would be anticipated that a sensitivity of at least 0.99 could be achieved, as this requires only an increase of 0.1% with rounding over the achieved sensitivity of Rodriguez-Meira. See MPEP 2144.05(II).
Double Patenting
Claims 16 and 18-37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-45 of copending Application No. 18/861,541 in view of Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025), Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.), and Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to a method comprising contacting a single cell with lysis buffer comprising at least one primer; adding to the lysis buffer a neutralization buffer, at least one polymerase, and a mixture of nucleotides comprising a terminator nucleotide that terminates amplification; amplifying at least some of a genome (claim 1); and sequencing the amplicons (claim 21).
The method of ‘541 recites that the concentration of magnesium is less than 8 mM (claim 16) and the composition of ‘541 recites that the magnesium is 2-4 mM (claim 29).
‘541 recites that the sequencing results in one or more of:
an allelic balance of at least 0.8 (claim 22)
a 1x coverage of at least 0.95 (claim 22)
a precision of at least 0.99 (claim 22)
an SNV sensitivity of at least 0.85 (claim 22)
‘541 teaches that the lysis buffer comprises the components of instant claim 22 (claim 2) and instant claim 22 (claim 3) at the concentrations of instant claim 24 (claim 4) at the pH of instant claim 25 (claim 5). ‘541 teaches the terminator nucleotide concentration (claim 20) and the dNTP concentrations (claims 18-19).
‘541 teaching the timings of claims 17-18 and 21 (claims 6-8) and that the method comprises ERAT and/or ligation (claim 9).
‘541 teaches analyzing 100 cells (claim 23).
In teaching concentrations of magnesium of 2 mM and dNTPs of 2mM, the composition teaches a ratio of at least one dNTP to magnesium of 1 (claims 29 and 33).
‘541 fails to teach the narrower allelic balance, 1x coverage, precision, and SNV sensitivity limitations and explicitly teach the lack of purification step(s).
As described and cited in the 103 rejection above, Gawad teaches:
an allelic balance capable in such a method of 1.0 (instant claim 29);
a lack of purification steps (instant claim 20);
Gawad teaches that alkaline lysis can degrade RNA and denature the genome (para [0083]). Gawad teaches that there is a need for highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods for research, diagnostics, and treatment involving small samples (para [0002]), and that its methods facilitate highly accurate amplification of target nucleic acids that increase accuracy and sensitivity of downstream applications including sequencing (para [00066]).
Gawad teaches that its methods allow for amplification with more uniform and reproducible coverage at lower error rates (para [00146]) and that modifications including adding exonuclease-resistant primers to the lysis buffer result in improved amplification uniformity over MDA (para [00154]).
As described and cited in the 103 rejection above, Gawad and Takara teach or suggest:
the precision (instant claim 31)
Takara teaches that its method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2) and that the workflow is economical (pg. 1, para 3).
As described and cited in the 103 rejection above, Gawad and Rodriguez-Meira teach or suggest:
the SNV sensitivities (instant claims 32-33)
Rodriguez-Meira teaches that its method of single cell sequencing dramatically increases the sensitivity of mutation detection (pg. 1293, Results, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the methods of ‘541 with the methods of Gawad, Dean, Takara, and Rodriguez-Meira as discussed above, motivated by the desire to improve the accuracy/sensitivity/amplification uniformity in a scalable and straightforward manner, as taught by Gawad, Takara, and Rodriguez-Meira. In doing so, at least the metrics claimed would have been obvious to assess and optimize for as discussed prior. There would have been a strong expectation of success as all are directed to nucleic acid amplification techniques for small amount of starting material (e.g., single cells) and/or MDA-based amplification.
Where the compositions of ‘541 are taught, such would be obvious for use in the method of ‘541 to one of ordinary skill in the art to utilize such a composition in the method as it is taught for the same purpose and/or as reference for optimization within the range taught by the method. See MPEP 2144.05 and 2144.06.
The optimized variables are likewise optimizable for the same reasons identified in the 103 rejections above.
Any additional limitations of the co-pending claims are encompassed by the open claim language “comprising” found in the instant claims.
Claims 16 and 18-37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 4-5, 11-18, 19, 23, 26, 34-39, and 42-43 of copending Application No. 17/631,130 in view of Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025), Saxonov (WO 2012/149042 A2), Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.), and Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to a method of analysis of genomic DNA from a sample comprising a single cell, the method comprising lysis of the single cell (claim 20) and contacting the genome with an amplification primer, a polymerase, a mixture of nucleotides including at least one terminator nucleotide, amplifying at least some of the genome to generate at least one terminated amplification product, ligating adapters, and sequencing the amplicons (claim 1).
‘130 teaches that the at least one terminated amplification product comprises at least 97% of the single cell’s genome (claim 15), wherein the artisan would recognize that a sequencing allowed to be of any (i.e., up to infinite) depth could capture the same percentage of coverage.
‘130 teaches sequencing both cDNA and the genome (claim 1) and detecting mutations (e.g., claims 22-23).
‘130 fails to teach or explicitly teach: specific limits of allelic balance, precision, SNV sensitivity, and timing of steps; buffers and components/concentrations thereof; a number of cells analyzed; that the lysis buffer is added prior to the neutralization buffer/that the primer is added at a separate time to the polymerase and nucleotides; or a lack of purification steps.
As described and cited in the 103 rejection above, Gawad teaches:
a lysis and a neutralization buffer, wherein the primer is added with the lysis buffer and polymerase and nucleotides are added subsequent to the neutralization (instant claim 1);
an allelic balance capable in such a method of 1.0 (instant claims 1 and 29);
a lack of purification steps (instant claim 20);
timing of adding of the lysis buffers and amplification (instant claims 1, 17-18, and 20);
a number of cells that may be analyzed (instant claim 26);
a concentration of terminator nucleotides (instant claim 28).
Gawad teaches that alkaline lysis can degrade RNA and denature the genome (para [0083]). Gawad teaches that there is a need for highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods for research, diagnostics, and treatment involving small samples (para [0002]), and that its methods facilitate highly accurate amplification of target nucleic acids that increase accuracy and sensitivity of downstream applications including sequencing (para [00066]).
Gawad teaches that its methods allow for amplification with more uniform and reproducible coverage at lower error rates (para [00146]) and that modifications including adding exonuclease-resistant primers to the lysis buffer result in improved amplification uniformity over MDA (para [00154]).
As described and cited in the 103 rejection above, Gawad and Saxonov teach or suggest:
the concentration of magnesium, dNTPs, ratios of dNTP(s) to magnesium (claims 16, 35-37).
Saxonov teaches methods for nucleic acid analysis (title), including single cell genomic sequencing comprising MDA (para [00176-00179]), wherein said amplification may use phi29 and occur within partitions/droplets for performing an amplification reaction (para [00134]). Saxonov teaches that such multiplexing is useful for evening out biological variation (para [00236]).
As described and cited in the 103 rejection above, Gawad, Saxonov, and Dean teach or suggest:
that amplification may be performed within an hour (claim 21);
the buffering agents; lysis buffer component concentrations of 10-500 mM; and pH (instant claims 22-25)
Dean also teaches a lack of purification steps, wherein direct amplification from blood or cultured cells after treatment with a base without need to physically separate DNA is taught as advantageous (col 6, para 1), wherein alkaline lysis can cause less damage to genomic DNA and thus result in higher quality DNA than other methods of lysis and wherein neutralization does not reactivate protein factors that can cause interference with amplification (col 36, para 2).
Dean teaches that the methods allow amplification of target nucleic acids from whole genomes and highly complex nucleic acid samples (col 8, Detailed Description, para 1), that the genome can be amplified through simple cell lysis techniques and amplification performed on crude lysates (col 6, para 1), and that the method generates plentiful amounts of amplified DNA that faithfully reproduces the locus representation frequencies of the starting material (col 42, para 5).
As described and cited in the 103 rejection above, Gawad and Takara teach or suggest:
the precision (instant claim 31)
Takara teaches that its method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2) and that the workflow is economical (pg. 1, para 3).
As described and cited in the 103 rejection above, Gawad and Rodriguez-Meira teach or suggest:
the SNV sensitivity of at least 0.99 (instant claims 32-33)
Rodriguez-Meira teaches that its method of single cell sequencing dramatically increases the sensitivity of mutation detection (pg. 1293, Results, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the methods of ‘539 with the methods of Gawad, Saxonov, Dean, Takara, and Rodriguez-Meira as discussed above, motivated by the desire to improve the accuracy/sensitivity/amplification uniformity in a scalable and straightforward manner, as taught by Gawad, Saxonov, Dean, Takara, and Rodriguez-Meira. In doing so, at least the metrics claimed would have been obvious to assess and optimize for as discussed prior. There would have been a strong expectation of success as all are directed to nucleic acid amplification techniques for small amount of starting material (e.g., single cells) and/or MDA-based amplification.
The optimized variables are likewise optimizable for the same reasons identified in the 103 rejections above.
Any additional limitations of the co-pending claims are encompassed by the open claim language “comprising” found in the instant claims.
Claims 16 and 18-37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-2, 4, 7, 9, 11, 13-15, 17-18, 21, 24, 28, 35-38, 42, and 47 of copending Application No. 18/860,539 in view of Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025), Saxonov (WO 2012/149042 A2), Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.), and Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to a method of amplifying genomic DNA from a single cell by contacting the genomic DNA with an amplification primer, a polymerase, a mixture of nucleotides including at least one terminator nucleotide, and sequencing the amplicons (claim 1).
‘539 teaches that the genomic DNA library comprises an allelic balance of up to 95% (claim 35) and an SNV sensitivity of 85% (claim 36).
‘539 teaches sequencing both cDNA and the genome (claim 1).
‘539 fails to teach or explicitly teach: buffers and components/concentrations thereof including a concentration of magnesium; all specific limits of coverage, precision, SNV sensitivity, and timing of steps; a number of cells analyzed; that the cell is lysed and that the lysis buffer is added prior to the neutralization buffer/that the primer is added at a separate time to the polymerase and nucleotides; ligation/ERAT; or a lack of purification steps.
As described and cited in the 103 rejection above, Gawad teaches:
a lysis and a neutralization buffer, wherein the primer is added with the lysis buffer and polymerase and nucleotides are added subsequent to the neutralization (instant claim 1);
a lack of purification steps (instant claim 20);
lysis and timing of adding of the lysis buffers and amplification (instant claims 1, 17-18, and 20);
treatment with end repair and A-tailing after step (c) (instant claim 19);
a number of cells that may be analyzed (instant claim 26);
a concentration of terminator nucleotides (instant claim 28).
Gawad teaches that alkaline lysis can degrade RNA and denature the genome (para [0083]). Gawad teaches that there is a need for highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods for research, diagnostics, and treatment involving small samples (para [0002]), and that its methods facilitate highly accurate amplification of target nucleic acids that increase accuracy and sensitivity of downstream applications including sequencing (para [00066]).
Gawad teaches that its methods allow for amplification with more uniform and reproducible coverage at lower error rates (para [00146]) and that modifications including adding exonuclease-resistant primers to the lysis buffer result in improved amplification uniformity over MDA (para [00154]).
As described and cited in the 103 rejection above, Gawad and Saxonov teach or suggest:
the concentration of magnesium, dNTPs, ratios of dNTP(s) to magnesium (claims 16, 35-37).
Saxonov teaches methods for nucleic acid analysis (title), including single cell genomic sequencing comprising MDA (para [00176-00179]), wherein said amplification may use phi29 and occur within partitions/droplets for performing an amplification reaction (para [00134]). Saxonov teaches that such multiplexing is useful for evening out biological variation (para [00236]).
As described and cited in the 103 rejection above, Gawad and Dean teach or suggest:
the buffers/solutions, components, and concentrations (instant claims 22-25)
Dean also teaches a lack of purification steps, wherein direct amplification from blood or cultured cells after treatment with a base without need to physically separate DNA is taught as advantageous (col 6, para 1), wherein alkaline lysis can cause less damage to genomic DNA and thus result in higher quality DNA than other methods of lysis and wherein neutralization does not reactivate protein factors that can cause interference with amplification (col 36, para 2).
Dean teaches that the methods allow amplification of target nucleic acids from whole genomes and highly complex nucleic acid samples (col 8, Detailed Description, para 1), that the genome can be amplified through simple cell lysis techniques and amplification performed on crude lysates (col 6, para 1), and that the method generates plentiful amounts of amplified DNA that faithfully reproduces the locus representation frequencies of the starting material (col 42, para 5).
As described and cited in the 103 rejection above, Gawad and Takara teach or suggest:
the precision (instant claims and 31)
Takara teaches that its method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2) and that the workflow is economical (pg. 1, para 3).
As described and cited in the 103 rejection above, Gawad and Rodriguez-Meira teach or suggest:
the SNV sensitivities (instant claims 32-33)
Rodriguez-Meira teaches that its method of single cell sequencing dramatically increases the sensitivity of mutation detection (pg. 1293, Results, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the methods of ‘539 with the methods of Gawad, Saxonov, Dean, Takara, and Rodriguez-Meira as discussed above, motivated by the desire to improve the accuracy/sensitivity/amplification uniformity in a scalable and straightforward manner, as taught by Gawad, Dean, Takara, and Rodriguez-Meira. In doing so, at least the metrics claimed would have been obvious to assess and optimize for as discussed prior. There would have been a strong expectation of success as all are directed to nucleic acid amplification techniques for small amount of starting material (e.g., single cells) and/or MDA-based amplification.
The optimized variables are likewise optimizable for the same reasons identified in the 103 rejections above.
Any additional limitations of the co-pending claims are encompassed by the open claim language “comprising” found in the instant claims.
Claims 16 and 18-37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-19 and 21-31 of copending Application No. 18/882,493 in view of Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025), Saxonov (WO 2012/149042 A2), Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.), and Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to contacting a sample comprising gDNA (claim 6) with a primer, a polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises a terminator nucleotide; and amplifying at least one nucleotide to generate a plurality of amplicons (claim 1), wherein the amplicons are sequenced (claim 8). See also claim 17.
‘493 teaches the concentration of alpha-thio dideoxy nucleotides [at least one terminator nucleotide] is 250-1200 uM.
The claims of ‘493 fails to teach or explicitly teach: all specific limits of coverage, precision, allelic balance, and timing of steps; buffers and components/concentrations thereof, including concentrations of magnesium; a number of cells analyzed; that the cell is lysed and that the lysis buffer is added prior to the neutralization buffer/that the primer is added at a separate time to the polymerase and nucleotides; ligation/ERAT; or a lack of purification steps.
MPEP 804 recites:
“In construing the claims of the reference patent or application, a determination is made as to whether a portion of the specification, including the drawings and claims, is directed to subject matter that is within the scope of a reference claim. For example, assume that the claim in a reference patent is directed to a genus of compounds, and the application being examined is directed to a species within the reference patent genus. If the reference patent discloses several species within the scope of the reference genus claim, that portion of the disclosure should be analyzed to properly construe the reference patent claim and determine whether it anticipates or renders obvious the claim in the application being examined. Because that portion of the disclosure of the reference patent is an embodiment of the reference patent claim, it may be helpful in determining the full scope and obvious variations of the reference patent claim.”
Therefore, it is further identified that specification of ‘493 identifies the species that read on the instant claims, wherein the applicant could have claimed such species in the original application:
‘493 para [0005] and [0007] recite an embodiments identifying low frequency sequencing variants, wherein the low frequency sequence variants constituted 0.01% of the total sequences. The artisan would understand that such would encompass a sequencing result of SNV sensitivity of about 0.99.
‘493 Fig. 5A recites detection of a fraction of genomic coverage of about 0.98. ‘493 para [0075] describes embodiments directed at coverage uniformity wherein no more than 50% of a cumulative fraction comprises sequences of at least 90% of a cumulative fraction of the target molecule. At least given the distributions of Fig. 5A, the artisan would expect coverages of 0.05 or greater in the remaining 50% cumulative fraction.
As described and cited in the 103 rejection above, Gawad teaches:
an allelic balance (instant claims 1 and 29);
a lysis and a neutralization buffer, wherein the primer is added with the lysis buffer and polymerase and nucleotides are added subsequent to the neutralization (instant claim 1);
a lack of purification steps (instant claim 20);
lysis and timing of adding of the lysis buffers and amplification (instant claims 1, 17-18, and 20);
treatment with end repair and A-tailing after step (c) (instant claim 19);
a number of cells that may be analyzed (instant claim 26);
coverage of the genome (instant claims 1 and 30);
a concentration of terminator nucleotides (instant claim 28).
Gawad teaches that alkaline lysis can degrade RNA and denature the genome (para [0083]). Gawad teaches that there is a need for highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods for research, diagnostics, and treatment involving small samples (para [0002]), and that its methods facilitate highly accurate amplification of target nucleic acids that increase accuracy and sensitivity of downstream applications including sequencing (para [00066]).
Gawad teaches that its methods allow for amplification with more uniform and reproducible coverage at lower error rates (para [00146]) and that modifications including adding exonuclease-resistant primers to the lysis buffer result in improved amplification uniformity over MDA (para [00154]).
Gawad teaches combined mRNA and gDNA analysis, wherein the DNA in the single cells is effectively amplified using the combined protocol (para [00203]; see also Example 4).
As described and cited in the 103 rejection above, Gawad and Saxonov teach or suggest:
the concentration of magnesium, dNTPs, ratios of dNTP(s) to magnesium (claims 16, 35-37).
Saxonov teaches methods for nucleic acid analysis (title), including single cell genomic sequencing comprising MDA (para [00176-00179]), wherein said amplification may use phi29 and occur within partitions/droplets for performing an amplification reaction (para [00134]). Saxonov teaches that such multiplexing is useful for evening out biological variation (para [00236]).
As described and cited in the 103 rejection above, Gawad and Dean teach or suggest:
the buffers/solutions, components, and concentrations (instant claims 22-25)
Dean also teaches a lack of purification steps, wherein direct amplification from blood or cultured cells after treatment with a base without need to physically separate DNA is taught as advantageous (col 6, para 1), wherein alkaline lysis can cause less damage to genomic DNA and thus result in higher quality DNA than other methods of lysis and wherein neutralization does not reactivate protein factors that can cause interference with amplification (col 36, para 2).
Dean teaches that the methods allow amplification of target nucleic acids from whole genomes and highly complex nucleic acid samples (col 8, Detailed Description, para 1), that the genome can be amplified through simple cell lysis techniques and amplification performed on crude lysates (col 6, para 1), and that the method generates plentiful amounts of amplified DNA that faithfully reproduces the locus representation frequencies of the starting material (col 42, para 5).
As described and cited in the 103 rejection above, Gawad and Takara teach or suggest:
the precision (instant claim 31)
Takara teaches that its method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2) and that the workflow is economical (pg. 1, para 3).
As described and cited in the 103 rejection above, Gawad and Rodriguez-Meira also teach or suggest:
the SNV sensitivities (instant claims 32-33)
Rodriguez-Meira teaches that its method of single cell sequencing dramatically increases the sensitivity of mutation detection (pg. 1293, Results, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the methods of ‘493 with the methods of Gawad, Saxonov, Dean, Takara, and Rodriguez-Meira as discussed above, motivated by the desire to improve the accuracy/sensitivity/amplification uniformity in a scalable and straightforward manner, as taught by Gawad, Dean, Takara, and Rodriguez-Meira. In doing so, at least the metrics claimed would have been obvious to assess and optimize for as discussed prior. There would have been a strong expectation of success as all are directed to nucleic acid amplification techniques for small amount of starting material (e.g., single cells) and/or MDA-based amplification.
It further would have been understood by the artisan, at least given the embodiments of application of ‘493, that the sequencing of those claims could result in the genomic coverage and/or SNV sensitivity claimed, and further would have been obvious to achieve such results in view of Gawad and/or Rodriguez-Meira for the reasons discussed and taught above.
Any additional limitations of the co-pending claims are encompassed by the open claim language “comprising” found in the instant claims.
Claims 16 and 18-37 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 11,905,553 B2 in view of Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025), Saxonov (WO 2012/149042 A2), Rodriguez-Meira (Rodriguez-Meira A, et al. Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing. Mol Cell. 2019 Mar 21;73(6):1292-1305.e8. Epub 2019 Feb 12.), and Takara (Takara Bio Blog Team. Takara Bio. 2019 [cited 2026 Feb 7]. Available from: https://catalog.takara-bio.co.jp/PDFS/accurate-detection-of-snvs-and-cnvs-from-5-cell-inputs.pdf).
Both sets of claims are directed to amplifying a target nucleic acids, which may be gDNA (claim 20) and obtained from a single cell (claim 19), comprising contacting a sample with at least one amplification primer, at least one polymerase, and a mixture of nucleotides comprising a terminator nucleotide; amplifying the at least one target nucleic acid (claim 1); and sequencing (claim 16).
‘553 fails to teach or explicitly teach: all specific limits of coverage, precision, allelic balance, and timing of steps; buffers and components/concentrations thereof; a number of cells analyzed; that the cell is lysed and that the lysis buffer is added prior to the neutralization buffer/that the primer is added at a separate time to the polymerase and nucleotides; ligation/ERAT; or a lack of purification steps.
MPEP 804 recites:
“In construing the claims of the reference patent or application, a determination is made as to whether a portion of the specification, including the drawings and claims, is directed to subject matter that is within the scope of a reference claim. For example, assume that the claim in a reference patent is directed to a genus of compounds, and the application being examined is directed to a species within the reference patent genus. If the reference patent discloses several species within the scope of the reference genus claim, that portion of the disclosure should be analyzed to properly construe the reference patent claim and determine whether it anticipates or renders obvious the claim in the application being examined. Because that portion of the disclosure of the reference patent is an embodiment of the reference patent claim, it may be helpful in determining the full scope and obvious variations of the reference patent claim.”
Therefore, it is further identified that specification of ‘553 identifies the species that read on the instant claims, wherein the applicant could have claimed such species in the original application:
‘553 col 5, lines 24-28 recite an embodiments identifying low frequency sequencing variants, wherein the low frequency sequence variants constituted 0.01% of the total sequences. The artisan would understand that such would encompass a sequencing result of SNV sensitivity of about 0.99.
‘553 Fig. 5A recites detection of a fraction of genomic coverage of about 0.98. ‘553 col 1, Brief Summary, para 1 describes embodiments directed at coverage uniformity wherein no more than 50% of a cumulative fraction comprises sequences of at least 90% of a cumulative fraction of the target molecule. At least given the distributions of Fig. 5A, the artisan would expect coverages of 0.05 or greater in the remaining 50% cumulative fraction.
As described and cited in the 103 rejection above, Gawad teaches:
an allelic balance (instant claims 1 and 29);
a lysis and a neutralization buffer, wherein the primer is added with the lysis buffer and polymerase and nucleotides are added subsequent to the neutralization (instant claim 1);
a lack of purification steps (instant claim 20);
lysis and timing of adding of the lysis buffers and amplification (instant claims 1, 17-18, and 20);
treatment with end repair and A-tailing after step (c) (instant claim 19);
a number of cells that may be analyzed (instant claim 26);
coverage of the genome (instant claims 1 and 30); and
a concentration of terminator nucleotides (instant claim 28).
Gawad teaches that alkaline lysis can degrade RNA and denature the genome (para [0083]). Gawad teaches that there is a need for highly accurate, scalable, and efficient nucleic acid amplification and sequencing methods for research, diagnostics, and treatment involving small samples (para [0002]), and that its methods facilitate highly accurate amplification of target nucleic acids that increase accuracy and sensitivity of downstream applications including sequencing (para [00066]).
Gawad teaches that its methods allow for amplification with more uniform and reproducible coverage at lower error rates (para [00146]) and that modifications including adding exonuclease-resistant primers to the lysis buffer result in improved amplification uniformity over MDA (para [00154]).
Gawad teaches combined mRNA and gDNA analysis, wherein the DNA in the single cells is effectively amplified using the combined protocol (para [00203]; see also Example 4).
As described and cited in the 103 rejection above, Gawad and Saxonov teach or suggest:
the concentration of magnesium, dNTPs, ratios of dNTP(s) to magnesium (claims 16, 35-37).
Saxonov teaches methods for nucleic acid analysis (title), including single cell genomic sequencing comprising MDA (para [00176-00179]), wherein said amplification may use phi29 and occur within partitions/droplets for performing an amplification reaction (para [00134]). Saxonov teaches that such multiplexing is useful for evening out biological variation (para [00236]).
As described and cited in the 103 rejection above, Gawad and Dean teach or suggest:
the buffers/solutions, components, and concentrations (instant claims 22-25)
Dean also teaches a lack of purification steps, wherein direct amplification from blood or cultured cells after treatment with a base without need to physically separate DNA is taught as advantageous (col 6, para 1), wherein alkaline lysis can cause less damage to genomic DNA and thus result in higher quality DNA than other methods of lysis and wherein neutralization does not reactivate protein factors that can cause interference with amplification (col 36, para 2).
Dean teaches that the methods allow amplification of target nucleic acids from whole genomes and highly complex nucleic acid samples (col 8, Detailed Description, para 1), that the genome can be amplified through simple cell lysis techniques and amplification performed on crude lysates (col 6, para 1), and that the method generates plentiful amounts of amplified DNA that faithfully reproduces the locus representation frequencies of the starting material (col 42, para 5).
As described and cited in the 103 rejection above, Gawad and Takara teach or suggest:
the precision (instant claims and 31)
Takara teaches that its method that combines high-quality library prep with amplicon-based enrichment to detect targeted SNVs, wherein the libraries can be prepped in a single day reducing turnaround time and labor (pg. 1, para 1-2) and that the workflow is economical (pg. 1, para 3).
As described and cited in the 103 rejection above, Gawad and Rodriguez-Meira teach or suggest:
the SNV sensitivities (instant claims 32-33)
Rodriguez-Meira teaches that its method of single cell sequencing dramatically increases the sensitivity of mutation detection (pg. 1293, Results, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed inventions to have combined the methods of ‘553 with the methods of Gawad, Saxonov, Dean, Takara, and Rodriguez-Meira as discussed above, motivated by the desire to improve the accuracy/sensitivity/amplification uniformity in a scalable and straightforward manner, as taught by Gawad, Dean, Takara, and Rodriguez-Meira. In doing so, at least the metrics claimed would have been obvious to assess and optimize for as discussed prior. There would have been a strong expectation of success as all are directed to nucleic acid amplification techniques for small amount of starting material (e.g., single cells) and/or MDA-based amplification.
It further would have been understood by the artisan, at least given the embodiments of patent of ‘553, that the sequencing of those claims could result in the genomic coverage and/or SNV sensitivity claimed, and further would have been obvious to achieve such results in view of Gawad and/or Rodriguez-Meira for the reasons discussed and taught above.
Any additional limitations of the co-pending claims are encompassed by the open claim language “comprising” found in the instant claims.
Response to Arguments
Applicant’s arguments, see pg. 8-10, filed 05/22/2026, with respect to the concentration of magnesium have been fully considered and are persuasive. The 102 rejection as anticipated by Gawad of claims 16, 18-20, 22-23, 26-29, and 34; the 103 rejection of claims 16-23, 26-29, and 34 over Gawad; and the non-statutory double patenting of ‘130, ‘539, ‘541, ‘493, and ‘553 have been modified in view of the amendments.
The claims did not previously require a particular level of magnesium. New rejections have been made incorporating the teachings of Saxonov, as necessitated by amendments, which supplies the necessary teachings regarding magnesium in view of at least Gawad.
Applicant's arguments filed 05/22/2026 regarding the non-statutory double patenting of ‘541 have been fully considered but they are not persuasive.
Applicant's arguments fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Namely, the claims of ‘541 recite a concentration of magnesium. The rejection has been updated accordingly.
Conclusion
No claims are 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Emma R Hoppe whose telephone number is (703)756-5550. The examiner can normally be reached Mon - Fri 11:00 am - 7:00 pm.
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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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