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 .
Priority
This application claims priority to provisional application PRO 63/225,081 filed 07/23/2021. All claims will be given the earlier filing date.
Claim Status
Claims 1-21 are pending and under examination. Claims 1 and 21 are independent claims.
Claim Objections
Claims 3, 6, 12, 15, 16 are objected to because of the following informalities: typographical errors.
Claim 3 recites “…wherein the first plurality bonding sites of the surface comprises non-covalent bonding sites” and should recite “…wherein the first plurality of bonding sites of the surface comprise non-covalent bonding sites” to maintain proper antecedent back to claim 1 and use the correct grammar.
Claim 6 recites “…wherein the first plurality bonding sites…” and should recite “…wherein the first plurality of bonding sites…”
Claim 12 recites “…and the second plurality of the bonding sites of the surface;” should read “…and the second plurality of bonding sites of the surface;”.
Claim 15 recites “…wherein the second plurality bonding sites on the surface comprises comprise covalent bonding sites.” The claim should read “… wherein the second plurality of bonding sites of the surface comprise covalent bonding sites.”
Claim 16 recites “…wherein the second plurality bonding sites…” and should recite “…wherein the second plurality of bonding sites…”Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
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 1-20 are 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.
The term “about” in claims 1, 9, 10, and 12 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. In regards to claims 1 and 12, the total salt concentration used is ambiguous as claim 1 requires “about 100 mM or less” while claim 12 requires “about 250 mM or greater.” The use of the term “about” renders the scope of these limitations unclear because the claims do not provide any objective boundaries for determining permissible ranges encompassed by “about.” Similarly in regards to claim 10, the use of “about” renders to the upper limit of buffer pH ambiguous.
In regards claim 9, the term “about” is used to describe each endpoint of the range of polynucleotide concentrations, “about 10 pM to about 2000 pM, about 100 pM to about 1000 pM, about 200 pM to about 500 pM, or about 250 pM to about 350 pM” as such it is unclear what the metes and bounds of the claimed polynucleotide concentration. Furthermore, claim 9 recites a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) which may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 9 recites the broad recitation of “about 10 pM to about 2000 pM”, and the claim also recites ranges of “about 100 pM to about 1000 pM, about 200 pM to about 500 pM, or about 250 pM to about 350 pM” which are narrower statements of the range/limitation. The claim is considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-21 are rejected under 35 U.S.C. 103 as being unpatentable over Lin et al. (US 2012/0316086 A1, published Dec. 13, 2012, on IDS 10/14/2022) in view of Gao et al. (Nucleic Acids Research, published 2006) and Bowen et al. (US 2013/0116153 A1, published May 9, 2013, on IDS 10/14/2022).
In regards to claims 1 and 12, Lin teaches methods for preparing sequencing substrates comprising contacting a solution comprising template polynucleotides with a surface of a substrate having bonding sites for capturing nucleic acids and attaching the template polynucleotides to the surface via covalent or non-covalent interactions (see Lin [0040]-[0044], [0060]-[0068]). Lin further teaches that such substrates comprise functionalize surfaces having immobilized oligonucleotides that serve as primers for sequencing reactions thereby providing multiple bonding sites for nucleic acid interactions, including two or more populations of bonding sites (see Lin [0060]-[0068], [0073]). Thus, Lin teaches “a method of preparing a substrate for sequencing, comprising: contacting a first buffer solution comprising template polynucleotides with a surface of the substrate, wherein the surface wherein the surface of the substrate comprises a first plurality of bonding sites … and a second plurality of bonding sites; and attaching the template polynucleotides to the surface of the substrate by forming covalent bonding or non-covalent bonding between the template polynucleotides and first plurality of bonding sites of the surface. ”
Bowen likewise teaches sequencing substrates comprising functionalized surfaces configured for attachment of nucleic acids, including template molecules and primer oligonucleotides, via covalent or non-covalent bonding interactions with functional groups (see Bowen [0079], [0089], [0101]). Bowen further teaches that such substrates include arrays of discrete sites for localized nucleic acid interactions, supporting the use of multiple bonding sites and attachment of different nucleic acid species at the substrate surface (see [0067]).
Thus, Lin and Bowen teach “a method of preparing a substrate for sequencing, comprising: contacting a first buffer solution comprising template polynucleotides with a surface of the substrate.” Furthermore, Lin teaches using two populations of bonding sites (see [0073]) reading “wherein the surface wherein the surface of the substrate comprises a first plurality of bonding sites … and a second plurality of bonding sites; and attaching the template polynucleotides to the surface of the substrate.”
However, neither Lin nor Bowen explicitly disclose that the template polynucleotides are contacted with the substrate using buffer solution comprising a total salt concentration of about 100 mM or less, for claim 1, nor a buffer solutions comprising a total salt concentration of about 250 mM or greater, for claim 12.
Gao teaches that nucleic acid hybridization and binding behavior depend on solution conditions including ionic strength and buffer composition, and demonstrates hybridization under varying salt concentrations, thereby evidencing that salt concentration affects nucleic acid hybridization and surface interactions (see Gao pg. 3370 lt. col. 1st para., pg. 3371, rt. Col. 2nd para., pg. 3373 rt. Col. 3rd para.). Because nucleic acid hybridization and attachment are governed by solution conditions, a person of ordinary skill in the art would have recognized that salt concentration represents a result effective variable that may be adjusted to control nucleic acid binding, hybridization efficiency, and surface loading.
With respect to claim 1, it would have been obvious to employ a first buffer solution having a selected salt concentration of about 100 mM or less to facilitate controlled hybridization and attachment of template polynucleotides to the substrate surface as a matter of routine optimization of ionic conditions in view of Gao.
With respect to claim 12, Lin teaches that oligonucleotides are contacted with the surface of the sequencing substrate and immobilized to serve as primers for sequencing reactions (see Lin [0060]-[0068]), and Bowen further reinforces attachment of oligonucleotides to functionalized surfaces (see Bowen [00034]-[0038], [0045]-[0048]). Sequencing flow-cell systems such as those described by Lin and Bowen ordinarily involve the sequential introduction of multiple reagent solutions, including solutions containing template nucleic acids and solutions containing primer oligonucleotides, as part of routine sequencing workflows recognized by those of ordinary skill in the art. Because salt concentration influences nucleic acid hybridization and binding interactions, one of ordinary skill in the art would have recognized that the salt concentration of the buffer used during primer attachment likewise represents a result-effective variable that may be adjusted to promote efficient attachment or hybridization. Accordingly selecting a second buffer solution having a salt concentration of about 250 mM or greater would have been an obvious matter of routine optimization of ionic conditions in order to facilitate primer attachment and surface binding.
Additionally, both Lin and Bowen are directed to sequencing substrate systems employing surface-bound nucleic acids for sequencing reactions, and it would have been obvious to combine their teachings to implement known surface chemistries and substrate configurations in order to improve nucleic acid capture, localization, and sequencing performance. Accordingly, the methods of claims 1 and 12 would have been obvious over the combined teachings of Lin, Bowen, and Gao.
In regards to claim 2, Lin teaches using single-stranded primers as the template polynucleotide (see Lin [0052]-[0055]). Lin teaches that nucleic acid templates introduced into sequencing flow cells are typically single-stranded nucleic acids that hybridize to complementary oligonucleotides immobilized on the surface of the sequencing substrate (see Lin [0045]-[0048], [0052]-[0055]). Sequencing-by-synthesis reactions performed on flow-cell substrates require single-stranded templates so that complementary primers can hybridize and polymerase extension can occur from the primer-binding site (see Lin [0060]-[0063]). While Bowen teaches the use of either single- or double-stranded DNA (see Bowen [0074])
In regards to claims 3-5, Lin teaches that the bonding sites of the surface comprise non-covalent bonding sites, including affinity binding and the use of streptavidin and biotin binding pairs (see Lin [0050]). Likewise, Bowen teaches using either covalent or non-covalent linkages (see Bowen [0079], [0086], [0089]-[0098]), and teaches the use of streptavidin and biotin binding pairs (see Bowen [0078]-[0079], [0107], [0111]).
In regards to claims 6-8 and 15-17, Lin teaches sequencing substrates in which nucleic acids are immobilized on functionalized surfaces through chemical attachment reactions between reactive functional groups on the surface and complementary reactive groups on the nucleic acid or linker molecules used for immobilization, including binding surface comprise covalent bonding sites (see Lin [0072]), wherein the covalent bonding sites comprise amino bonding sites (see Lin [0101]), azido bonding sites or NHS ester moieties (see Lin [0102]-[0103]). While Bowen teaches a wide variety of functional groups including aminos, azides, and click-chemistry groups, and thiol bonding (see Bowen [0077]-[0081]). It was well known in the art that biomolecules such as nucleic acids may be immobilized on solid substrates using a wide variety or reactive functional groups including amino, carboxyl, thiol, aldehyde, azide, alkyne, and related click-chemistry groups, as well as enzyme-mediated ligation systems such as sortase-based coupling and protein tag systems (e.g., SNAP-tag or CLIP-tag). These functional groups represent well-known alternative coupling chemistries used to covalently attach biomolecules to functionalized surfaces, many of which are taught by Bowen (see 0079], [0086], [0089]-[0098], and throughout). It therefore would have been obvious to one of ordinary skill in the art at the time of filing to employ any of the recited functional groups as bonding sites on the sequencing substrate in order to covalently attach nucleic acid molecules to the surface as a matter of routine selection among known chemical coupling strategies for biomolecule immobilization.
In regards to claim 9, Lin teaches introducing nucleic acid templates into a sequencing flow cell where the templates hybridize to complementary oligonucleotides immobilized on the surface (see Lin [0052]-[0060]). The concentration of nucleic acid templates used in such hybridization reactions is a parameter that directly affects hybridization efficiency and surface occupancy. Gao teaches that nucleic acid hybridization kinetics depend on factors such as ionic strength, temperature, and concentration of nucleic acids and performs hybridization reactions using controlled nucleic acid concentrations to evaluate duplex formation kinetics (see Gao pg. 3372 rt. Col. 2nd para., pg. 3375 rt. Col. 2nd para. ). It would have been obvious to one of ordinary skill in the art to select a suitable concentration of template polynucleotides within the claimed picomolar range as a matter of routine optimization of a result-effective variable in order to control hybridization efficiency and surface loading of nucleic acid molecules on the sequencing substrate. The claimed range of about 10 pM to about 2000 pM represents a typical concentration range used for nucleic acid hybridization reactions and does not produce any unexpected results relative to the teachings of the prior art. Accordingly, the additional limitations of claim 9 would have been obvious for the same reasons set for the rejection of claim 1.
In regards to claim 10, Lin teaches introducing nucleic acid templates into a sequencing flow cell where the templates hybridize to complementary oligonucleotides immobilized on the surface (see Lin [0052]-[0060]). Gao likewise teaches that nucleic acid hybridization behavior is affected by experimental conditions including differences in ionic strength, pH, strand sequence and concentration, temperature, and buffer additives (see Gao pg. 3375 rt. Col. 2nd para.) A person of ordinary skill in the art would have recognized that the pH of the buffer solution represents a result-effective variable that can be adjusted to control nucleic acid hybridization behavior, stability of nucleic acids, and surface interactions during template capture. Selecting a pH value within the claimed range of about 3.5 or less therefore would have been an obvious matter of routine optimization of buffer conditions in order to obtain suitable hybridization or template capture behavior in the sequencing system taught by Lin. Accordingly, the additional limitation of claim 10 would have been obvious for the same reasons set forth in the rejection of claim 1.
In regards to claim 11, depends on claim 1 and further recites that “the first buffer solution further comprises one or more crowding agents.” Macromolecular crowding agents such as polyethylene glycol (PEG) are well known in the art for user in nucleic acid hybridization and enzymatic reactions involving nucleic acids because such agents increase the effective concentration of macromolecules in solution and promote intermolecular interactions influencing nucleic acid hybridization. Bowen teaches using PEG to encourage molecular crowding and concentrate template molecules leading to enhanced rates of capture (see Fig. 34, [0051], [0107]). A person of ordinary skill in the art would have recognized that adding a crowding agent such as PEG to the buffer solution used during template hybridization in the sequencing method of Lin would increase the efficiency of nucleic acid interactions and surface capture of template molecules. Because crowing agents such as PEG were well known additives in nucleic acid reaction buffers used to enhance hybridization and related reactions, selecting a buffer that further comprises one or more crowding agents would have been an obvious modification of the buffer conditions used in the method of Lin as a matter of routine optimization of reaction conditions.
In regards to claim 13 and 14, Bowen teaches sequencing substrates comprising immobilized oligonucleotides that serve as primers for amplification and sequencing reactions in a flow-cell environment, including methods using multiple types of primer oligonucleotides with distinct primer sequences immobilized of the surfaces to enable amplification and sequencing of nucleic acid templates, including forward and revers primer sequences that correspond to adaptor sequences on the template molecules such as the combination of P5 and P7 paired end primers (see Bowen [0112], [0115]). It was well known in the art at the time of the invention that sequencing by synthesis systems, including those used in flow-cell platforms utilize paired adaptor-specific primer sequences (e.g., P5 and P7 sequences) to facilitate cluster amplification and sequencing of template nucleic acids. Accordingly, selecting primer oligonucleotides comprising P5 and P7 primer sequences represents the use of known, standard sequencing primers in the sequencing system taught by Lin and would have been an obvious design choice for one of ordinary skill in the art.
In regards to claim 18, Lin expressly teaches that the nucleic acid templates immobilized on a sequencing substrate are amplified on the surface to generate clusters of clonal copies of the template polynucleotides for sequencing (see [0052], [0073]).
In regards to claim 19, the claim depends on claim 1 and further recites that “the surface of the substrate comprises a plurality of patterned nanowells.” While Lin teaches sequencing substrates comprising functionalized surfaces for attachment of nucleic acids and subsequent amplification, including patterned surfaces (see Title, Abstract, throughout) and that the surfaces may be in wells of a multiwell plate (see [0032], [0042], [0046]-[0047]), Lin does not expressly teach using a nanowell format.
Bowen however, teaches an alternative substrate architecture for sequencing systems of the type taught by Lin, comprising arrays of discrete, patterned reaction sites including wells having sub-micron dimensions (e.g., less than 1 µm, such as approximately 30-500 nm, or openings of about 100 nm2) configured to localize nucleic acids molecules at defined positions on the substrate surface (see [0082], [0095], [0103]). Such structures constitutes patterned nanowells that confine nucleic acid molecules within discrete locations and enable controlled loading and sequencing of nucleic acid templates. It would have been obvious to one of ordinary skill in the art to modify the sequencing substrate of Lin to include patterned nanowell structures taught by Bowen in order to improve spatial organization of template molecules, reduce cluster overlap, and enhance sequencing accuracy and signal resolution. Furthermore, one would expect a high likelihood of success as both references are directed to sequencing substrate technologies employing related surface chemistries and architecture.
In regards to claim 20, Bowen teaches that the patterned sites are configured to enable capture of single nucleic acid molecules per site, thereby promoting formation of a single template or dominant clonal population at each site, which corresponds to the claimed single-cluster occupancy of nanowells (see Title, [0067], [0069], [0111]). Achieving such occupancy levels represents an expected result of controlling template loading and confinement within discreate wells and would have been an obvious outcome of implementing the patterned nanowell structures taught by Bowen.
In regards to claim 21, Lin teaches substrates having surface bound nucleic acids for sequencing and Bowen teaches substrates comprising patterned nanoscale wells configured for localized nucleic acid capture and sequencing. Accordingly, the substrate of claim 21 represents a combination of known sequencing substrates and known patterned nanowell architectures and would have been obvious to one of ordinary skill in the art.
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681