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 . Applicant’s preliminary amendment filed 10/4/2024 is acknowledged. Claims 1-50 have been canceled. Claims 51-70 have been added and are pending.
Priority
This application is a CON of PCT/US2023/060778 filed 01/17/2023 which claims benefit of 63/300,343 filed 01/18/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/04/2024 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings were received on 7/3/2024. These drawings are found acceptable by the Examiner.
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) 51-70 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hannon et al (WO-2021116715, June 2021, effective filing date December 2019) in view of Choi et al (WO 2020021084, January 2020).
The claims are directed to a method of preparing nucleic acid molecules for sequencing, comprising: contacting double-stranded nucleic acid molecules attached to a surface with ethylene carbonate to generate single-stranded nucleic acid molecules attached to the surface; and, hybridizing sequencing primers to the single-stranded nucleic acid molecules, thereby generating sequencing hybrids.
Regarding claims 51 and 65, Hannon et al teach preparation of a solid surfaced labeled with a detection probe. In order to produce a double-stranded end on the detection probe, the BALI_06 oligonucleotide was diluted to a final 1 mM concentration in hybridization buffer (10% ethylene carbonate in 2X SSC) and incubated on the slide surface for 15 minutes at room temperature, followed by two 5’ washes in hybridization solution at room temperature and three washes in 2X SSC at room temperature (page 53, beginning at line 21 to page 54, line 15). Hannon et al teaches that BALI_07 and BALI_08 oligos were mixed to a 5 mM final concentration in 2X SSC buffer, heated at 95°C for 5 minutes, and allowed to cool down at room temperature for 30 minutes. A ligation solution was prepared by mixing: 107.5 pi of ultra-pure water, 125 ml 2x quick ligation mix (NEB), 12.5 ml T4 ligase, high concentration (NEB), and 5 mI (final 100uM) of BALI_07/08 oligos. The ligation solution was incubated on the slide for 30 minutes at room temperature, followed by three 5’ washes in 2X SSC. After the first series of washes, the slide was imaged again using the same parameters of the first imaging, only in the cy5 channel (page 53, beginning at line 21 to page 54, line 15). Hannon teaches further teaches wherein the detection probe may further comprises one or more sequencing elements, wherein the sequencing element is a primer and wherein the primer is used for sequencing library amplification (page 21, lines 12-16)
Regarding 52, Hannon teaches wherein the ethylene carbonate has a concentration of between about 10% volume/volume (page 53).
Regarding claim 53, Hannon teaches wherein the incubation is at room temperature which is about 37oC (page 53).
Regarding claim 54, Hannon teaches wherein the nucleic acids of the method may encompass sequences comprising a peptide nucleic acid (PNA) (page 12, line 29, to page 13, line 4).
Regarding claim 56, Hannon teaches wherein the sequencing element may encompass an adapter that is capable of functioning as a sequencing primer (page 6, lines 17-23).
Regarding claim 57, Hannon teaches wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are amplification products (pages 8 and 9).
Regarding claim 58, Hannon teaches covalent bonding and modification of the oligonucleotides (pages 19-21).
Regarding claim 59, Hannon teaches wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are attached to the surface using amine-reactive crosslinker chemistry (BS(PEG) crosslinker at page 51).
Regarding claim 60, Hannon teaches wherein the solid surface comprises of magnetic beads (page 46, figure 9 and Example 6) and wherein the substrate may be a glass or plastic or etc (page 4).
Regarding claim 61, Hannon teaches further comprising, prior to the contacting with ethylene carbonate (i) attaching nucleic acid molecules in a sequencing library to the surface and (ii) amplifying the nucleic acid molecules in the sequencing library attached to the surface, thereby generating sequencing colonies comprising the double-stranded nucleic acid molecules attached to the surface (e.g., pages 8-9).
Regarding claims 62-64, Hannon teaches wherein the nucleic acid molecules may comprise of amplification, wherein the amplification may encompass rolling circle amplification and/or using strand displacement polymerase, which inherently encompasses isothermal conditions and temperatures (page 9, lines 1-6, pages 39-40).
Regarding claim 65, Hannon teaches further comprising washing the nucleic acid molecules with a wash buffer (see e.g., Examples, especially Example 3 which discuss hybridization solution as wash buffer and wash solution comprising 2X SSC).
Regarding claim 66, Hannon teaches further comprising sequencing the single-stranded nucleic acid molecules, thereby generating sequencing data (last paragraph at page 3).
Regarding claims 67- 70, Hannon teaches wherein the single-stranded nucleic acid molecules are sequenced using a plurality of sequencing flow steps, each sequencing flow step comprising contacting the sequencing hybrids with nucleotides, wherein at least a portion of the nucleotides are labeled, and detecting the presence or absence of an incorporated nucleotide (See examples, esp. Ex 9 which discuss sequencing used to measure how many detection probes targeting GFP/RFP were present in each spatially barcoded population).
While Hannon teaches various embodiments throughout which overlaps with the instant invention including the use of ethylene carbonate in hybridization buffers and wash buffers for use in preparing nucleic acids for sequencing-based assay, Hannon does not expressly teach generating sequencing hybrids as claimed.
Regarding claim 51-70, Choi teaches a method of enriching for target nucleic acids to be sequenced by a single molecule sequencing by synthesis, the method comprising the steps of: a. isolating nucleic acids in a sample solution; b. conjugating the nucleic acids to adaptors, wherein the adaptors comprise universal primer binding sites and sequencing primer binding sites; c. amplifying the adapted target nucleic acids with universal primers to form target amplicons; d. contacting the sample with a formamide-free hybridization solution comprising one or more single-stranded hybridization probes linked to a binding moiety and further comprising a solvent selected from dimethyl sulfoxide (DMSO), sulfolane, ethylene carbonate, pyrrolidone or a primary amide; e. incubating the sample under conditions facilitating formation of hybrids between the target amplicons and the probes; f. isolating the hybrids by capturing the binding moiety; g. releasing amplicons from the hybrids (see claims).
Choi teaches sequencing workflow and further teaches a non-toxic alternative to formamide, which may include ethylene carbonate. Choi teaches that a suitable replacement has the properties of facilitating denaturation and increasing hybridization specificity. In addition, the presence of the formamide replacement may not interfere with any downstream applications and is suitable for next-generation nucleic acid sequencing applications.
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to utilize ethylene carbonate in the sequencing method of Hannon as taught by Choi et al with a reasonable expectation of success for the benefit of enriching target nucleic acid with increased specificity as suggested by Choi.
Conclusion
9. No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681
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