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 .
Election/Restrictions
Applicant’s election without traverse of Group II, claims 16-20 in the reply filed on 01/13/2026 is acknowledged. The claims of Group I have been cancelled.
Status of Claims
Applicant’s amendment filed 01/13/2026 is acknowledged. Claims 21-35 have been added. Claims 1-15 have been cancelled. Claims 16-35 are pending in the instant application and the subject of this non-final office action.
Priority
The instant application claims priority to provisional applications 63/338,669 and 63/406,862. These applications lack support for at least the numeric limits of the following limitation of claim 16:
“(d) sequencing the plurality of amplification products or amplicons thereof, wherein the sequencing results in one or more of:
i. an allelic balance of at least 0.8;
ii. a 1X coverage of at least 0.95;
iii. a precision of at least 0.99; and
iv. an SNV sensitivity of at least 0.85.”
Therefore, the claims, which all depend from claim 16, have been given a priority date of 05/04/2023 based on the PCT/US2023/021073.
Specification
The use of the terms including “Tween”, “Triton”, “Trizma”, “PicoPlex”, “Deep Vent”, “Therminator”, “KAPA”, and “Qubit”, each which is a trade name or a mark used in commerce, has been noted in this application. Such terms should be accompanied by the generic terminology; furthermore, such terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The disclosure is objected to because of the following informalities:
The text of the bodies of Tables 10A and 10B appears faint, and some digits are hard to read. If text was color or grey, Applicant should provide in black text to prevent issues during conversion.
Appropriate correction is required.
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 1, the term “a 1x coverage” in the phrase “a 1x coverage of at least 0.95" was interpreted to encompass “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 § 112(b)
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.
Claims 16-35 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 16, the claim recites “(a) contacting … a single cell with … amplification primer; … (c) amplifying at least some of the genome to generate … amplification products”.
There is insufficient antecedent basis for the limitation “the genome” in the claim. While a cell inherently comprises a genome, the claim also reads on methods that encompass, for example, artificial chromosomes (e.g., YACs and BACs) that may comprise exogenous “genome”. Likewise, the claims, which recite “comprising”, encompass samples with more than one cell. Therefore, it is not clear which genome is being referred to.
Claims 17-35 are indefinite for depending from claim 16 and not rectifying the deficiency.
Regarding claims 17-18 and 21, step (b) recites “adding to the lysis buffer…”. The examples (e.g., Examples 1-4) recite mixing, vortexing, and incubation times, but not “dispensing” or “adding” times, for example. Therefore, it is not clear whether the limitations directed to the length of time assigned to “step (b)”, for example, are intended to limit the time allotted to the dispensing of the neutralization buffer, the polymerase, and the nucleotides or, more broadly, any intervening time between step (a) and step (c), for example.
Further, the separation in timing between steps (b) and (c) is unclear as the methods encompass isothermal amplification methods and polymerases that may operate at some level of efficiency immediately after addition. For example, while the steps are not temperature-limited, phi29 is known to function at room temperature and would be expected to begin amplifying immediately under standard conditions.
For this reason, the metes and bounds are not clear to one of ordinary skill in the art.
Regarding claim 23, first, the claim recites “the buffering agent”. There is insufficient antecedent basis for this limitation in the claim.
Second, the claim contains the trademark/trade name “Trizma”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe “Tris(hydroxymethyl)aminomethane” (the base itself, the acid salt thereof, and/or a combination thereof) and, accordingly, the identification/description is indefinite.
Regarding claim 28, the claim recites “the at least one terminator nucleotide is present at a concentration of less than 0.3 mM”. It is not clear, in the case of more than one terminator nucleotide, whether the collective concentration of “the at least one terminator nucleotide” is intended to be less than 0.3 mM or if each terminator nucleotide of “the at least one terminator nucleotide” may be less than 0.3 mM.
Therefore, the metes and bounds are not clear to one of ordinary skill in the art.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 16, 18-20, 22-23, 26-29, and 34 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Gawad (WO 2021/022085 A2; published 02/04/2021; as cited in the IDS dated 11/03/2025).
Regarding claim 16, 20, 27-29, 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 at equal ratios of 1200 uM (para [00155]; instant claims 27-28). See also para [00185].
amplifying [genomic] DNA from the single cell (para [00155]; see also para [00160] which shows alignment to hg19, i.e., genomic)
sequencing the amplification products (para [00158-00164]), wherein the sequencing results in at least:
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 Gawad para [00128]; instant claim 29).
A 1x genomic coverage in cells analyzed by the method of 97% (Table 2; see also para [00204-205] and Fig. 11).
An SNV sensitivity of about 0.85 (Fig. 5E)
Gawad teaches no intervening purification steps among (a)-(c) (para [00154-155], [00185]; instant claim 20).
Regarding claims 18, Gawad teaches an embodiment in which denaturing Gawad recites adding quenching buffer, vortexing, centrifuging briefly, adding amplification mix, and incubating for 8 hours, at which point the amplification was terminated by heating (para [00154-155]).
For the purposes of compact prosecution, it is noted that Gawad teaches incubating the single cell on ice for 10 minutes (para [00154-155]).
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 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)).
Claim 23 has been interpreted as depending from claim 16 given the lack of antecedent basis to claim 16, and it is noted that claim 23 does not require the buffer to comprise a buffering agent.
Regarding claim 26, Gawad teaches analyzing at least 100 cells (para [0097]).
For at least these reasons, claims 16, 18-20, 22-23, 26-29, and 34 are anticipated by Gawad.
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.
Claim(s) 16-23, 26-29, and 34 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).
Regarding claim 16, 20, 27-29, 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 at equal ratios of 1200 uM (para [00155]; instant claims 27-28). See also para [00185].
amplifying [genomic] DNA from the single cell (para [00155]; see also para [00160] which shows alignment to hg19, i.e., genomic)
sequencing the amplification products (para [00158-00164]), wherein the sequencing results in at least:
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 Gawad para [00128]; instant claim 29).
A 1x genomic coverage in cells analyzed by the method of 97% (Table 2; see also para [00204-205] and Fig. 11)
An SNV sensitivity of about 0.85 (Fig. 5E)
Gawad teaches no intervening purification steps among (a)-(c) (para [00154-155], [00185]; instant claim 20).
Regarding claim 17 and 21, in the method of Gawad, Gawad teaches an embodiment in which cells are incubated 10 (para [00154-155]) to 25 minutes between a lysis and amplification (para [00169]). Broadly interpreted, step (b) may encompass an incubation time after contacting with the lysis buffer. Gawad teaches alkaline lysis to denature the genome (para [0083]) and that combinations of strategies may be used (para [00122]).
Therefore, it would have been obvious to optimize the length of the time at which the lysis buffer, polymerase, and nucleotide mixture are added based on the desired amount of lysis/denaturation and combination (or lack thereof) of techniques utilized for lysis, as taught by Gawad. See MPEP 2114.05(II).
Regarding claims 18, in the method of Gawad, Gawad teaches an embodiment in which denaturing Gawad recites adding quenching buffer, vortexing, centrifuging briefly, adding amplification mix, and incubating for 8 hours, at which point the amplification was terminated by heating (para [00154-155]).
For the purposes of compact prosecution, it is noted that Gawad teaches incubating the single cell on ice for 10 minutes (para [00154-155]).
Regarding claim 19, in the method of Gawad, Gawad teaches following amplification with addition of end repair and A-tailing (ERAT) mixture (Fig. 2A and 6; para [00158])
Regarding claim 22-23, in the method of Gawad, 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)).
Claim 23 has been interpreted as depending from claim 16 given the lack of antecedent basis to claim 16, and it is noted that claim 23 does not require the buffer to comprise a buffering agent.
Regarding claim 26, in the method of Gawad, Gawad teaches analyzing at least 100 cells (para [0097]; see also [00205]).
It is noted that the claim does not require whether the cells are in separate containers or in a single container when contacted with the lysis buffer.
Regarding claim 30, in the method of Gawad, Gawad teaches analyzing sequences in bulk to achieve higher genome coverage of at least about 0.98 (Fig. 5A). Gawad teaches that analysis of cells in bulk provides general information about the cell population but may be unable to detect low-frequence mutants over the background (para [0082]). Gawad teaches utilizing various numbers of cells in the methods (para [0097]).
Thus, the coverage at a depth of 1x (at least one read) is determined to be a routinely optimizable variable, dependent on at least the number of cells sequenced and combined for analysis, as taught by Gawad. See MPEP 2144.05(II).
Claim(s) 18 and 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) as applied to claim 16 above, and in view of Dean (US 7,074,600 B2; published 07/11/2006).
Regarding claims 18 and 21, in the method of Gawad and Dean, 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, 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 dNTP concentrations (para [00185]).
However, Gawad fails to teach specific concentrations of buffer components, lysis buffer H+ (i.e., pH), or dNTPs.
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 multiple concentrations of dNTPs utilized (entire document, for example the following): 100 uM (col 100, line 35), 200 uM dNTPs (col 100, line 13), 400 uM (col 92, line 56), and 1.0 mM (col 98, line 9). Thus, the concentration of dNTP is interpreted to be an optimizable variable, dependent on at least the chosen polymerase and amplification method.
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 with the buffer components, component concentrations, and the range of dNTPs utilize 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 both are directed to alkaline lysis of cell(s) and MDA-based methods.
Further, such buffer component and dNTP 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) as applied to claim 16 above, and 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, 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 fails to teach a sensitivity [sample variant concordance; see instant Table 9] of at least 0.995.
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).
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 panel method of Takara, motivated by the desire to reduce time, labor, and costs, as taught by Takara, as well as to improve the sensitivity. 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.
Claim(s) 32-33 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) as applied to claim 16 above, and 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 32-33, in the method of Gawad, 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 fails to teach an SNV sensitivity of 0.95 or 0.99.
Rodriguez-Meira rectifies this by teaching 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 0.01% with rounding over the achieved sensitivity of Rodriguez-Meira. See MPEP 2144.05(II).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 16-35 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), wherein 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). 5’41 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).
‘541 fails to teach the narrower allelic balance, 1x coverage, precision, and SNV sensitivity limitations and explicitly teach the lack of purification step.
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 Dean teach or suggest:
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 ‘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, 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 16-35 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), 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 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, 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 16-35 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), 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: all specific limits of coverage, precision, SNV sensitivity, 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.
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 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, 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 16-35 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), 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; 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 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 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, 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 16-35 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), 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 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, 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.
Conclusion
No claims are allowed.
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/EMMA R HOPPE/ Examiner, Art Unit 1683
/ANNE M. GUSSOW/ Supervisory Patent Examiner, Art Unit 1683