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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/4/2025 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 1-3, 10, 17, 20, 24-25, 93-95, and 97-101 are currently pending. Claims 90-92 are currently withdrawn.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 8/5/2025 and 11/4/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Response to Applicant Amendments
Claim Objections
Claims 97 and 98 were objected to for various informalities. In light of Applicant’s amendments to the claims submitted 11/4/2025, these objections have been withdrawn. However, see new grounds of objection below.
35 USC 103 Rejections
Claims 1-3, 10, 17, 20, 24-25, 93-95, 97-101 were rejected as being unpatentable over Rothberg et al. (US 2010/0137143 A1) in view of Meyer et al. (Nucleic Acids Research, 2007) and various combinations of references. In light of Applicant’s amendments to the claims submitted 11/4/2025, these rejections have been withdrawn, and see “Response to Applicant’s Arguments” below. See also new grounds of rejection below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that Rothberg, the primary reference used in the Final Rejection, does not add nucleotides to an array in a template-independent manner, as required by the amended instant claims. Applicant also argues that the ordinary artisan would not be motivated to modify Rothberg to develop probes in this manner, because Rothberg is generally directed to sequencing methods, and so template-independent sequence creation would render the reference inoperable for its intended purpose and change its principle of operation (Remarks, pages 8-9).
The Examiner agrees that Rothberg does not contemplate template-independent sequence synthesis, which is now required in instant claim 1. Meyer does not remedy this deficiency. Thus, the previous 35 USC 103 Rejections have been withdrawn. The new grounds of rejection provided below do not incorporate Rothberg, and so Applicant’s arguments are rendered moot.
Claim Objections
Claim 1 is objected to because of the following informality: step (d) is unclear if a typographical error has occurred. In the final two lines of the step, it is stated that the concentration gradient of the second dNTP varies along a second direction from the first location, but the second dNTP is, at least initially, associated with a second location. It is unclear then if “from the first location” should read “from the second location.” It is noted that the current claim language does not render the scope of the claim unclear, and therefore does not qualify as a rejection under 35 USC 112(b). Appropriate correction is required.
Claim 2 is objected to because of the following informality: in line 1, “immobilized probe” should read “immobilized surface probe” to match similar language used throughout the claims. Appropriate correction is required.
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 1-3, 10, 17, 20, 24-25, 93-95, and 97-101 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.
Claim 1 is rejected because of the newly amended phrases describing first and second locations. Specifically, the array is exposed to a “concentration gradient” for the first and second dNTPs at a “first location” and a “second location,” respectively. It is unclear how a gradient would exist at the singular point of introduction of a dNTP on the array, nor how this concentration gradient at this location alone would result in nucleotides being added to the oligonucleotides of the immobilized probes. It is believed that Applicant’s intent is to clarify the origination point of each dNTP exposure on the array, where the concentration gradient for the array as a whole depends on said origination point, and said origination point differs between the first and second dNTPs. The claim will be interpreted as though this is the case, and Applicant is encouraged to amend the claim to read as such if this interpretation is correct.
Claims 2-3, 10, 17, 20, 24-25, 93-95, and 97-101 are rejected based on their dependence on rejected claim 1.
Claim Interpretation
With regard to the concentration gradient and the generation of the spatially barcoded array in claim 1, it is noted that page 254, para. 4 of the instant specification states, “The easiest way to create a concentration gradient is by diffusion. For example, a few drops of solution (e.g., nucleotide solution) can be added to a buffer, and the solute in the solution diffuse along the concentration gradient, from where the solute exists in its highest concentration (e.g., the site where the drop of solution is added) to where it occurs in its lowest concentration (e.g., the area where the solute has not reached by diffusion). In some embodiments, the diffusion will continue until the concentration of the solute becomes uniform in all directions of the buffer. Thus, this concentration gradient can be temporary. However, before the concentration of the solute becomes uniform, nucleotides can be readily added to the oligonucleotides, thus creating a spatial barcode based on the concentration gradient.” Therefore, a concentration gradient will be considered to be formed when a dNTP solution is flowed over an array from starting point which does not involve contact with all immobilized probes at the same time, and a spatially barcoded array will be considered to be made even when said concentration gradient is temporary, so long as the concentration gradient results in the addition of dNTPs to an existing immobilized oligonucleotide/probe.
Regarding claims 1 and 3, it is noted that at minimum, claim 3 only requires a single additional dNTP (as evidenced by the third or fourth dNTP language) to vary along a single direction (as evidenced by the third or fourth direction language) that is different from the “first direction” and “second direction” of claim 1. In the instant specification, it is stated that the first and second direction may be at a 90° or 180° angle from one another (page 3, para. 1), and therefore could be the direction of a flow and the direction directly against said flow. Additionally, neither claim 1 nor claim 3 prohibit the concentration gradient of a dNTP varying along multiple directions simultaneously.
Regarding claim 101, it is noted that one of the features of the plurality of features may simply be a single plurality of immobilized surface probes. As the other “features” are not specifically defined, any component of an array, or a component attached to an array, taught in the prior art will be considered a feature that reads on the instant 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.
Claims 1-3, 17, 20, 24-25, 95, 97-98, and 100-101 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (WO 2017/176541 A1).
Lee teaches methods for making polynucleotides via a template-independent polymerase and the repeated addition of nucleotides (Abstract). The template-independent polymerase used may be terminal deoxynucleotidyl transferase (TdT; page 3, para. 2; instant claim 17). The basic method is described in the joining para. of pages 3-4: a mobile reaction reagent with the template-independent polymerase and a selected dNTP (at a selected concentration) moves through a fluidic channel to a reaction site, where said reaction site has attached initiator sequences. The mobile reaction reagent has a particular volume and flow rate “to achieve a selected residence time at the reaction site” so as to add the dNTP to the initiator sequences. The reaction site is then washed, and the steps are repeated until polynucleotides are formed. As noted in Figures 2B and 4, multiple initiators immobilized to a surface may be used (see also page 7, para. 3 and page 9, para. 1 which note immobilization of the initiators to the surface). Four different dNTPs (such as dGTP, dTTP, dATP, and dCTP) may be flowed onto the initiators in a sequence, where the order of the dNTPs may be controlled by a user (page 9, para. 1 and page 27, para. 2; instant claim 95). As shown in Figures 2A and 4, in each cycling of each dNTP, one or more nucleotides are added to each initiator sequence (see also page 45, para. 2 instant claims 97-98). On page 10, para. 3, Lee also states, “The present disclosure provides that another important property of the mobile-phase synthesis strategy is that it allows a different condition to be used in each of the four TdT-dNTP packet types. This is important as the kinetics of the enzyme may be different for different dNTPs. Thus, to obtain optimal results, different conditions, such as type and concentration of divalent ions may need to be used for different dNTPs.” Thus, different concentrations may be used with each dNTP, which would therefore naturally form different concentration gradients (instant claim 100). It is noted that these concentration gradients vary in all directions relative to and from the initial location the dNTP enters the reaction site (see “Claim Interpretation” above).
The joining para. of pages 44 and 45 further notes the fluidic control of dNTPs, which can limit the time each dNTP is exposed to the initiators. Page 41, para. 3 notes that reagents can be delivered “at a desired location for a desired period of time.” This specific positioning/location delivering also applies to the microfluidic device embodiments of Lee (page 42, para. 1). Additionally, page 43, para. 2 states, “Reagents can be deposited onto a discrete region of the support, such that each region forms a feature of the array…If the angle of dispersion of reagents is narrow, it is possible to create an array comprising many features.” Lee also generally notes that reagents may be applied at “one or more locations” on a substrate containing the initiators (page 23, para. 2), stating on page 24, para. 2 that, “According to one aspect, the reagents may be added to one or more than one or a plurality of locations on the substrate in series or in parallel or the reagents may contact the entire surface of the support, such as by flowing the reagents across the surface of the support.” Page 25 , para. 2 notes that microfluidic channels may be used to access particular locations on a substrate. The initiator sequences may be attached to the array in a known fashion (page 23, para. 2).
Figures 6-7 of Lee also show that for increased concentrations of a nucleotide, there is generally increased extension of initiators. Lee also teaches the use of “addressable arrays” where the locations of initiator sequences are known (page 23, para. 2), and “addressable” locations are spatially defined (page 33, para. 1).
Thus, prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Lee to arrive at the methods of claims 1 and 3. Specifically, Lee clearly lays out extending single-stranded oligonucleotides via a terminal transferase and a cyclical flow of dNTPs with particular concentration gradients. Additionally, Lee states that multiple locations on the array can be used to disperse the reagent mixtures containing the dNTPs. Combining the teachings of Lee concerning concentration gradients, discrete locations for different reagent mixtures, and the concept of “addressable arrays” would lead the ordinary artisan to recognize that different dNTPs could be flowed onto the array at different discrete locations, and due to the natural concentration gradients formed in all directions from such a method, the initiator sequences would be extended differently depending on their location on the array and their distance from the introduction of each dNTP. Lee teaches that after extension is complete, the resulting nucleotides may be amplified and sequenced (page 15, para. 1). Thus, the ordinary artisan would be capable of looking at the sequencing data and determining where on the array a particular sequence originated. According to the instant specification, a "spatial barcode" is a “contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier that conveys or is capable of conveying spatial information,” (page 62, para. 2). Such a use of the methods of Lee would therefore naturally result in the creation of a spatial barcode. The ordinary artisan would be motivated to combine the teachings of Lee in such a manner because the resulting extended array has many practical uses, such as spatially labeling and analyzing biological samples or generating/testing/analyzing potential primer or adapter sequences. Lee also teaches that, “The methods according to the present disclosure can be used for synthesis of cheaper, more accurate and longer custom DNA sequences for various biochemical, biomedical, or biosynthetic applications.” Therefore, the general methods of Lee are an improvement on existing array creation methods, and the ordinary artisan would recognize that by combining the teachings of Lee as described above, these same benefits would apply to a spatially barcoded array.
Thus, claims 1, 3, 17, 95, 97-98, and 100 are prima facie obvious over Lee.
Regarding claims 2 and 24, Lee teaches that initial immobilization of nucleic acids to a solid support generally includes covalently attaching single stranded oligonucleotides (page 37, para. 2). Linker sequences can specifically be used, where the linker can include chemically reactive segments that are cleavable (page 38, para. 3). The reference also notes the use of a linker sequence that is common to all of the nucleic acids on said solid support (page 39, para. 3). The common linker sequence appears to be included in an embodiment of Lee that uses primers for initial attachment of nucleic acids to a solid support. However, it would be prima facie obvious to the ordinary artisan that a common, cleavable linker sequence may generally be used for the attachment of the initiator sequences to the solid support. Lee teaches that generally, attachment methods for nucleic acids on solid support, as well as methods involving linkers, are well-known in the art, providing a reasonable expectation of success (pages 37-38). The linkers can also be used to removed nucleic acids on the solid support, such as may be desired after extension is complete (page 38, para. 2). Thus, by including the same linker with the same structure on all of the initiator sequences attached to a substrate, it would allow for: 1) assurance that all the initiators/extended sequences will similarly remain attached to the substrate and 2) confidence that all of the extended sequences may be removed in the same manner. This would allow for a generally easier sequence removal process. Additionally, by making a linker sequence the same across all of the initiator sequences, during downstream analyses such as sequencing, filtering out these linker sequences would be easier, leading to more accurate and less noisy data.
Thus, claims 2 and 24 are prima facie obvious over Lee.
Regarding claim 20, in the rejection of claims 1 and 3 described above, the spatial barcode is generated from the natural concentration gradients formed from the dNTPs and their introduction into the reaction site from discrete locations. As four dNTPs are cyclically flowed onto the reaction site, it would be prima facie obvious that the spatial barcodes would involve all four dNTPs. By utilizing all of the dNTPs in this manner, it can ensure further spatial distinction between extended sequences in discrete locations on the array.
Regarding claim 25, Lee teaches that the microfluidic devices of their invention that deliver the reagents to the initiators may have multiple channels (page 25, para. 2, page 42, paras. 1-2).
Regarding claim 101, as noted above, a plurality of initiators may be used. Page 33, para. 1 notes that substrates may also have other features (such as pores or beads).
Claims 10, 93-94, and 99 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (WO 2017/176541 A1) in view of Belgrader et al. (US 2018/0179591 A1; cited in a previous Office Action).
Regarding claim 99, Lee teaches the method of claim 98, as described above. Lee also teaches, in their working examples, that following the synthesis of a sequence, a DNA adapter can be ligated to the end of the sequence via the use of a ligase (step 3 on page 52 and step 11 on page 58). However, the ligated sequences are not poly(T) sequences. Lee also generally notes that the immobilized probes of their invention may participate in hybridization (e.g. page 39, paras. 2-3), particularly for downstream analyses (page 54, para. 1). The reference also teaches that the sequences produced by their methods can be used for “biochemical, biomedical, or biosynthetic applications,” (page 11, para. 1).
Belgrader teaches that mRNAs contain poly(A) tail sequences (e.g. paras. 218, 257, and Figure 9). This sequence is at the 3’ end of the mRNA. In Figure 44, Belgrader shows the ligation of a poly(T) segment (reference character 4431) to a primer (see the top strand of reference character 4430) so that it may target the poly(A) of an mRNA (para. 431). This reference also teaches that ligation reactions can be done via ligase enzymes (para. 206). The reference also teaches that utilizing their methods can characterize biological and biochemical materials, and that particularly, applying their methods to sequencing can allow for the obtaining of information for diagnostics, prognostics, and biotechnology (e.g. paras. 3-5 and 230).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Belgrader to ligate a poly(T) oligonucleotide to the synthesized sequences of Lee, specifically for aiding in downstream hybridizing methodologies involving mRNA. This would entail extending the sequences on the supports of Lee as normal, and then ligating a poly(T) adapter to the sequences upon completion with a ligase, as is already done in Lee with non-poly(T) adapter sequences, and as is shown in Belgrader. By including this poly(T) sequence, the end of an mRNA can be hybridized to the synthesized sequence via the poly(A) tall, as is also shown in Belgrader. This is simply enacting a particular scenario for the stated applications of Lee guided by the teachings of Belgrader. The addition of the poly(T) sequence will allow the synthesized sequences to hybridize to mRNAs regardless of the content of the rest of their sequences, and by hybridizing to mRNAs specifically, this could allow for spatial analysis of biological samples (as is generally described in the rejections of claims 1 and 3 above) including cells or tissues. Such hybridization can also be used in the diagnostic, prognostic, and biotechnology methods described above by Belgrader. These line up well with the established “biochemical, biomedical, or biosynthetic applications” of Lee, and so the ordinary artisan would be capable of using ordinary creativity to combine the two references. There would be a reasonable expectation of success as Lee already teaches that their synthesized sequences can have adapters ligated via ligase, and Belgrader teaches hybridization of mRNAs to attached probes via poly(A) and poly(T) interactions.
Thus, claim 99 is prima facie obvious over Lee in view of Belgrader.
Regarding claims 10 and 93-94, as is noted above in the rejection of claims 1 and 3, Lee teaches that the order of the dNTPs flowed onto the array can be controlled. Page 9, para. 1 notes that the nature of the nucleotides added can be controlled, as does page 11, para. 2 and page 31, para. 2, for example. Example flow orders include “GATC” (page 9, para. 1), “GATACA” in Figure 1A, and “ACT” in Figures 2B and 4. These teachings render it prima facie obvious to the ordinary artisan that any order of dNTPs could be used in the method of Lee, particularly as the reference does not prohibit any such order.
Thus, given the teachings of Belgrader described above and the obviousness rationale that would be possible for the ordinary artisan to construct, it would also be prima facie obvious that instead of ligating a poly(T) oligonucleotide to the end of the synthesized sequences, dTTP could simply be used as the last dNTP flowed onto the reaction site. Lee teaches that their template-independent polymerases often create homopolymers for each dNTP introduced (see page 3, para. 3, page 23, para. 1, pages 23-24 joining para., Figure 2B, and Figure 4, where the figures show homopolymer sequences utilizing dTTPs specifically). By utilizing dTTPs to create a poly(T) sequence, it simplifies the overall method of Lee in view of Belgrader, as the step of adapter ligation is not required, while still obtaining the same result of the combination, as the poly(T) sequence created in this manner would still be capable of hybridizing to a poly(A) mRNA sequence. This would still confer the same downstream analysis benefit results of Lee in view of Belgrader (e.g. spatial barcoding of samples, diagnostic and prognostic value, etc.), and so would be motivating to the ordinary artisan.
Thus, claims 10 and 93-94 are prima facie obvious over Lee in view of Belgrader.
Conclusion
No claims are currently allowable.
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/F.F.G./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681