DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status/Action Summary
This action is in response to the papers filed on February 2, 2026.
Claims 105-107 and 109 were canceled in the response. Claims 99-104, 108, and 110-118 are under examination. No other claims are currently pending in the present application.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections of record regarding the absence of sequence identifiers in the description of the drawings, the language used in the abstract, and sequence listing disclosures have been withdrawn in view of the substitute specification and abstract filed with the response.
The rejections of record under 35 U.S.C. 112(b) have been withdrawn in view of the amendments to the claims.
The rejections of record under 35 U.S.C. 102 have been withdrawn in view of the amendments to independent claim 99 now requiring that the “…first single-stranded binding moiety compris[es] a single-stranded binding (SSB) protein…”
Priority
The present application was filed on February 24, 2023 and is a CON of PCT/US2021/048523, filed on August 31, 2021, which claims priority to U.S. Provisional Application No: 63/073305, filed on September 1, 2020.
Claim Interpretation
The claim term “cobalt hexammine” appears to be an art-recognized synonym for “cobalt hexamine”. National Center for Biotechnology Information. "PubChem Compound Summary for CID 159295, Hexaaminecobalttrichloride" PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/Hexaaminecobalt-trichloride. Accessed 26 September, 2025.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 99-102, 108, 110-116 are/remain rejected under 35 U.S.C. 103 as being unpatentable over McKernan et al., US 2009/0062129 A1, published March 5, 2009 in view of Daunay et al., “Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer” Nucleic Acids Research, Volume 47, No. 21, September 17, 2019.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 99, McKernan et al. teach methods of processing particles for massively parallel nucleic acid sequencing comprising providing particles coupled to nucleic acid molecules wherein “blocking oligos” (i.e. single-stranded binding moieties) are hybridized to the particle-coupled nucleic acids (McKernan et al., figure 36).
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McKernan et al. further teach clonal populations (of particle-immobilized nucleic acids) are present at discrete locations on the semi-solid support (i.e. the surface), wherein each clonal population comprises a tag so that the discrete location contains fragments containing 3’ and 5’ tags that can be independently sequenced (i.e. immobilizing treated particles to different individually addressable locations on a substrate (McKernan et al., paragraphs 0186-0188).
McKernan et al. do not teach that the “single-stranded binding moiety” comprises a single-stranded binding (SSB) protein.
However, Daunay et al. teach analysis of microsatellite loci (i.e. repetitive sequences) in genomic DNA is complicated by introduction of undesirable frameshift artifacts during PCR known as “stutter or shadow bands/peaks” following analysis by capillary electrophoresis or next generation sequencing (Daunay et al., page 2, column 1, paragraphs 1-2). Daunay et al. teach a method for isothermal amplification of genomic DNA comprising microsatellite repeats wherein single stranded DNA strands (i.e. single-stranded portions) are stabilized by single-stranded DNA binding proteins (Daunay et al., page 2, column 1, paragraphs 4-column 2, paragraph 1), resulting in “drastically reduce[d] formation of stutter artifacts” (Daunay et al., page 11, column 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of processing a plurality of particles for nucleic acid sequencing, taught by McKernan et al., comprising hybridizing blocking oligonucleotides (i.e. single-stranded binding moieties) to particle-immobilized nucleic acids (i.e. first nucleic acids immobilized to particles) with the teachings of Daunay et al. to utilize single-stranded DNA binding proteins to stabilize the single stranded portion of the particle-immobilized nucleic acid molecule rather than hybridizing “blocking oligonucleotides” thereto. The ordinary artisan would have been motivated to replace the “blocking oligonucleotides” taught by McKernan et al. with the single-stranded binding proteins taught by Daunay et al. because of the teaching of Daunay et al. that isothermal amplification of target nucleic acids (i.e. first nucleic acid molecules) comprising stabilizing single stranded templates drastically reduces the occurrence of stutter artifacts when amplifying genomic DNA comprising microsatellite repeats.
Regarding claim 100, Mckernan et al. teach the first nucleic acid molecule comprises a sequence of a sample nucleic acid molecule (the “unique DNA sequence”), and priming sequences (the “adapter” sequences) (McKernan et al., figure 36).
Regarding claim 101, McKernan et al. teach sequencing populations of nucleic acids wherein each microparticle comprises a unique primer binding region (i.e. the first nucleic acid molecule comprises a barcode sequence or a unique molecular identifier sequence) (McKernan et al., paragraph 0029).
Regarding claim 102, McKernan et al. teach hybridizing "blocking oligonucleotides" to the first nucleic acid molecule (i.e. providing a reaction mixture comprising single stranded binding moieties) (McKernan et al., figure 36 and paragraph 0081).
Regarding claim 108, McKernan et al. teach the first nucleic acids are captured on the beads by "capture primers" (i.e. the particle comprises immobilized nucleic acid molecules (the capture primers) with partial sequence identity to the first nucleic acid sequence) (McKernan et al., figure 35).
Regarding claim 110, McKernan et al. teach immobilizing the particles to a substrate (i.e. the solution comprising the particles is in contact with the substrate) (McKernan et al., paragraph 0030).
Regarding claim 111, McKernan et al. teach contacting a plurality of particles with a plurality of single-stranded binding moieties to yield a plurality of treated particles comprising different particles bound to the single-stranded binding moieties (McKernan et al. figure 36 and paragraph 0081) (also see McKernan et al., paragraph 0216).
Regarding claims 112-113, McKernan et al. teach microspheres or beads (i.e. particles) of a variety of diameters may be selected for the method, including those with diameters approximately 0.5 microns or less (i.e. a diameter less than 1 micrometer) (McKernan et al., paragraph 0098). McKernan et al. further teach that it is desirable to employ procedures to reduce clumping of the microparticles (McKernan et al., paragraph 0191), and that ideally the density of beads loaded onto a sequencing surface is low enough that the beads are not touching another bead such that the optical center-of-mass of each individual bead can be identified and a region around the bead integrated to compute the bead intensity… with a dark background around them (McKernan et al., paragraph 0431).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have recognized the presence and/or frequency of particle aggregates (two or more beads touching each other on the sequencing surface) as a parameter in the method taught by McKernan et al. that could be optimized by routine experimentation. The ordinary artisan would have been motivated to optimize the methods of McKernan et al. to minimize the frequency of particle aggregates because of the teaching of McKernan et al. that, in the most desirable case wherein the beads are not touching other beads (i.e. there are no aggregates present), analysis of the sequencing time-series of images is greatly simplified because each bead can be simply defined as a region of an expected size surrounded by a dark background (McKernan et al., paragraph 0431).
Regarding claim 114, McKernan et al. teach immobilizing the blocking oligonucleotide - hybridized particles to a substrate (McKernan et al., paragraphs 0030-0031).
Regarding claim 115, McKernan et al. teach denaturing double-stranded nucleic acid molecules "solution PCR products" prior to immobilizing the nucleic acid molecules to the beads (McKernan et al., figure 35).
Regarding claim 116, McKernan et al. teach sequencing the single-stranded nucleic acid molecules immobilized to the beads (McKernan et al., figure 35).
Response to arguments
The response asserts that the amended claim 99 is non-obvious over McKernan in view of Daunay because (1) the cited references do not meet all of the elements of claim 99 and (2) one of skill in the art would not be motivated to modify McKernan with Daunay because modification of McKernan with Daunay would render McKernan inoperable for its intended purpose.”
The response asserts: (1) McKernan in view of Daunay does not teach “contacting said first nucleic acid molecule with a first single-stranded binding moiety comprising a single-stranded binding (SSB) protein…to yield a first treated particle”.
This assertion has been thoroughly reviewed and is not persuasive. As detailed above, McKernan et al. in view of Daunay et al. teach methods of processing particles for massively parallel sequencing wherein the particles comprise single stranded nucleic acid portions, immobilizing said particles to different sites on a surface, generating clonal clusters comprising 5’ and 3’ tags wherein aberrant amplification is suppressed by contacting the particles with blocking oligonucleotides (McKernan et al.) or single strand binding proteins (Daunay et al.).
The response asserts: (2) The modification of McKernan with Daunay would render McKernan “unsatisfactory for its intended purpose” because the blocking oligos used by McKernan are sequence-specific (are targeted to known sites that are shared among target molecules), and the single-stranded binding proteins taught by Daunay do not have sequences specificity and would “prevent generation of usable sequencing results”.
This assertion has been thoroughly reviewed and is likewise not persuasive because Daunay et al. teach inclusion of SSBPs in sequencing reactions stabilizes single-stranded nucleic acid portions by preventing the formation of unwanted secondary structures between sequences that are highly repetitive (i.e. shared) within and among template molecules that result in polymerase “stutter” and mispriming events and improve the quality of sequences obtained from such repetitive templates.
Furthermore, Nikiforov et al., US 8603792 B2 (issued 2013), cited here only for evidentiary purposes in response to the assertion that inclusion SSBPs would be expected to render inoperable methods for sequencing, teach methods for massively parallel sequencing comprising nanoparticles conjugated to nucleic acids and “proteins known to enhance biomolecular activity… such as single stranded DNA Binding Protein” wherein “the presence of such proteins on the nanoparticle surface can help to reduce the number of biomolecules on the particle surface, enhance DNA synthesis and read length, and reduce the formation of biomolecule-nanoparticle conjugate aggregates (Nikiforov et al., column 52, line 54-column 53, line 14) and specifically that sequencing reactions can be performed in buffers supplemented with SSBP (Nikiforov et al., column 163, lines 40-48). Therefore, it appears that rather than rendering methods for sequencing inoperable, the ordinary artisan would have expected binding of SSBP to nanoparticles comprising nucleic acids and/or inclusion of SSBP to beneficially suppress the formation of unwanted secondary structures (i.e. hybridization of repetitive sequences) during amplification/sequencing.
Claims 103-104 are/remain rejected under 35 U.S.C. 103 as being unpatentable over McKernan et al., US 2009/0062129 A1, published March 5, 2009 in view of Daunay et al., “Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer” Nucleic Acids Research, Volume 47, No. 21, September 17, 2019, as applied to claims 99-102, 108, 110-116 above, and further in view of Ouameur et al., “Structural Analysis of DNA Interactions with Biogenic Polyamines and Cobalt(III)hexamine Studied by Fourier Transform Infrared and Capillary Electrophoresis” The Journal of Biological Chemistry Vol. 279, No. 40, page 42041-42054, July 28, 2004.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claims 103-104, McKernan et al. in view of Daunay et al. do not teach the [hybridization] reaction mixture comprises spermine or cobalt hexamine (i.e. “cobalt hexammine”).
However, Ouameur et al. teach cobalt hexamine and biogenic polyamines (including spermine) have the well-known property of stabilizing double-stranded DNA such that the melting temperature of a DNA duplex can be greatly increased in low salt solutions (Ouameur et al., page 42041, column 2, paragraphs 3-4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of processing a plurality of particles for nucleic acid sequencing, taught by McKernan et al. in view of Daunay et al., comprising binding particle-immobilized nucleic acids with SSBPs (as taught by McKernan et al. in view of Daunay et al), or hybridizing blocking oligonucleotides (i.e. single-stranded binding moieties) to particle-immobilized nucleic acids (i.e. first nucleic acids immobilized to particles) to further comprise cobalt hexamine or spermine in the hybridization reaction.
The ordinary artisan would have been motivated to make said modification with a reasonable expectation of success because of the teaching of Ouameur et al. that inclusion of cobalt hexamine or spermine in a hybridization mixture greatly increases the melting temperature (i.e. stability) of the resulting double-stranded nucleic acid, and therefore energetically favoring the hybridization of the blocking oligonucleotides and/or primers and/or capture probes to their corresponding target sequences.
Claims 117 and 118 are/remain rejected under 35 U.S.C. 103 as being unpatentable over McKernan et al., US 2009/0062129 A1, published March 5, 2009 in view of Daunay et al., “Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer” Nucleic Acids Research, Volume 47, No. 21, September 17, 2019.
This rejection has been updated as necessitated by amendments to the claims.
Regarding claim 117, McKernan et al. teach methods of processing a plurality of particles for massively parallel nucleic acid sequencing comprising providing particles coupled to nucleic acid molecules wherein “blocking oligos” (i.e. single-stranded binding moieties) are hybridized to the particle-coupled nucleic acids (McKernan et al., figure 36) (i.e. generating a solution comprising particles and particles coupled to single-stranded binding moieties).
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McKernan et al. further teach microspheres or beads (i.e. particles) of a variety of diameters may be selected for the method, including those with diameters approximately 0.5 microns or less (i.e. a diameter less than 1 micrometer) (McKernan et al., paragraph 0098). McKernan et al. further teach that it is desirable to employ procedures to reduce clumping of the microparticles (McKernan et al., paragraph 0191), and that ideally the density of beads loaded onto a sequencing surface is low enough that the beads are not touching another bead such that the optical center-of-mass of each individual bead can be identified and a region around the bead integrated to compute the bead intensity… with a dark background around them (McKernan et al., paragraph 0431).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have recognized the presence and/or frequency of particle aggregates (two or more beads touching each other on the sequencing surface) as a parameter in the method taught by McKernan et al. that could be optimized by routine experimentation. The ordinary artisan would have been motivated to optimize the methods of McKernan et al. to minimize the frequency of particle aggregates because of the teaching of McKernan et al. that, in the most desirable case wherein the beads are not touching other beads (i.e. there are no aggregates present), analysis of the sequencing time-series of images is greatly simplified because each bead can be simply defined as a region of an expected size surrounded by a dark background (McKernan et al., paragraph 0431).
Regarding claim 118, McKernan et al. do not teach that the “single-stranded binding moiety” comprises a single-stranded binding (SSB) protein.
However, Daunay et al. teach analysis of microsatellite loci (i.e. repetitive sequences) in genomic DNA is complicated by introduction of undesirable frameshift artifacts during PCR known as “stutter or shadow bands/peaks” following analysis by capillary electrophoresis or next generation sequencing (Daunay et al., page 2, column 1, paragraphs 1-2). Daunay et al. teach a method for isothermal amplification of genomic DNA comprising microsatellite repeats wherein single stranded DNA strands (i.e. single-stranded portions) are stabilized by single-stranded DNA binding proteins (Daunay et al., page 2, column 1, paragraphs 4-column 2, paragraph 1), resulting in “drastically reduce[d] formation of stutter artifacts” (Daunay et al., page 11, column 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of processing a plurality of particles for nucleic acid sequencing, taught by McKernan et al., comprising hybridizing blocking oligonucleotides (i.e. single-stranded binding moieties) to particle-immobilized nucleic acids (i.e. first nucleic acids immobilized to particles) with the teachings of Daunay et al. to utilize single-stranded DNA binding proteins to stabilize the single stranded portion of the particle-immobilized nucleic acid molecule rather than hybridizing “blocking oligonucleotides” thereto. The ordinary artisan would have been motivated to replace the “blocking oligonucleotides” taught by McKernan et al. with the single-stranded binding proteins taught by Daunay et al. because of the teaching of Daunay et al. that isothermal amplification of target nucleic acids (i.e. first nucleic acid molecules) comprising stabilizing single stranded templates drastically reduces the occurrence of stutter artifacts when amplifying genomic DNA comprising microsatellite repeats.
Response to arguments
The response asserts that amended claims 117 and 118 are not obvious over McKernan, Braunlin and Daunay because “the cited references, alone or in combination do not meet all of the elements of claim 117”, emphasizing the added limitation “generating a solution comprising said plurality of particles and said subset of said plurality of particles coupled to single-stranded binding moieties with no more than 1% of particles of said plurality of particles included in a particle aggregate”.
These assertions have been thoroughly reviewed and are not persuasive.
The “generating a solution” step is addressed in the updated rejection above.
The emphasized “no more than 1% of particles…” was previously rejected using a “routine optimization” rationale based upon the teachings of the cited prior art references. This rationale does not appear to be addressed in the remarks regarding claim 117.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Nikiforov et al., US 8603792 B2 (issued 2013).
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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