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
Claims 1, 3-22 are pending and under examination. Claim 2 has been cancelled. Claims 1, 21, and 22 are independent claims. Claims 1 and 22 have been amended.
Response to Arguments
Rejections Withdrawn
The rejection of claims 1-22 are rejected under 35 U.S.C. 103 as being unpatentable over Mok in further view of Nicol and Assarsson is withdrawn following the Applicants amendments and cancellation of claim 2.
New Rejections
Claim Rejections - 35 USC § 102
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1, 3-5, 9-11, 17-18 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Hacohen et al. (US 2023/0235394 A1, published Jul. 27, 2023, with priority to 63/0039,004 filed Jun. 15, 2020).
Hacohen teaches methods for nucleic acid sequencing, particularly preparing nucleic acids for sequencing. Hacohen’s methods are designed to increase efficiency, throughput, and/or yield of long range sequencing platforms (see Hacohen Abstract).
In regards to claim 1, Hacohen discloses a method of detecting DNA sequences from multiple pools, wherein each pool comprises at least one species of DNA molecule (see Hacohen [0007], [0102] disclosing “multiple families of these fragments can be made and processed to direct hybridization and subsequent ligation”). Hacohen discloses preparing pools wherein each pool has a defined end sequence which may be joined in a concatenation step, wherein the DNA molecules in the same pool have the same end sequences with different pools having different end sequences (see Hacohen [0102], Table 1 disclosing distinct adapter sequences, each one tied to distinct input population) and further discloses that “each adapter sequence possesses one or two designate sequence(s) that are complementary with at least one other of the plurality of nucleic acid sequences with an adapter sequence” (see Hacohen [0017]) reading on the limitation that “DNA molecules from one pool may only be joined to a DNA molecule from one or two pre-determined different pools.” Hacohen further discloses combining the pools to form a combination pool (see Hacohen [0095]-[0097]).
Hacohen further discloses generating at least one linear DNA concatemer wherein a position of each DNA molecule within the concatemer correlated to the pool from which the DNA molecule originate (see Hacohen Figs. 6, 7, and 9, [0082]-[0085] expressly demonstrating expected ligation order A->B ->C->D->…->P). Hacohen teaches sequencing the concatemers, thereby detecting the DNA sequences of each DNA molecule at each position (see Hacohen [0019], [0037]-[0038]) and discloses correlating the position of each DNA molecule within the concatemer to the pool from which the DNA molecule originated (see Hacohen [0045], [0213]).
Hacohen teaches all of the limitations of claim 1 in a single embodiment, as such the claim 1 is anticipated in its entirety by Hacohen and the claim is rejected under 35 U.S.C. 102.
In regards to claim 3, Hacohen teaches that nucleic acid sequences are amplicons generated in a DNA amplification reaction (see Hacohen [0012]).
In regards to claim 4, Hacohen cDNA fragments are DNA molecules whose sequence is specific for an analyte, and sequencing the DNA molecule results in detection of the corresponding analyte (original transcript) (see Hacohen [0037]-[0038]).
In regards to claim 5, Hacohen discloses generating multiple “families” (pools) by distinct multiplexed amplification reactions that simultaneously amplifies a defined set of target sequences using a family-specific primer pairs (see Hacohen Table 1, [0012], [0102]), a multiplex detection assay under BRI, since the reaction detects/amplifies multiple distinct targets simultaneously. Hacohen discloses performing this multiplex amplification separately for each of its eighteen discloses families each iteration yielding its own pool, thereby teaching multiple multiplex detection assays each yielding a pool.
In regards to claims 9-10, Hacohen discloses concatenation performed via “USER enzyme cocktail from NEB®)” (see Hacohen [0097], [0105]-[0108]). Hacohen further teaches Gibson assembly as an art-recognized alternative for forming the same linear array (see Hacohen [0097] disclosing “it is expressly contemplated that other routes for generating arrays could also be employed to make linear chimeric arrays, such as Gibson assembly”. Hacohen discloses the method comprises using assembly primer, wherein the primer pairs comprise unique assembly sites which is complementary to one unique assembly site in another, and wherein the PCR products of each pool are joined to the PCR products of different pools via their complementary assembly sites thereby generating the linear concatemers (see Hacohen Fig. 1, [0012], [0017]).
In regards to claim 11, Hacohen teaches using uracil-specific excision reagent assembly (see Hacohen Fig. 1, [0015]), and while Hacohen’s exemplified adapter structures shows a single dU per site in its working example, the disclosure specifically states “the instant disclosure employ specialized oligonucleotide primers designed to possess distinct complementary sequences that terminate at one or more dU residues and that can be used to prepare a linear tandem array of respective sequence elements”, thus Hacohen teaches using multiple uracil residues. Further it would have been obvious to a person of ordinary skill in the art to use multiple uracil residues within a single joining region to produce a single longer excised overhang from the same disclosed USER enzyme chemistry already taught by Hacohen, yielding the expected result of a longer, more discriminating single-stranded joining sequence (see MPEP 2143(A),(D)).
In regards to claims 17 and 18, Hacohen teaches methods of forming concatemers from DNA molecules originating from multiple pools as outlined above in the rejection of claim 1. Hacohen further teaches an index sequence that identifies which set of pools a given read derived from, disclosing that barcode sequences on nucleic acid probes may be shared across some or most, but not all, probes in a population, distinguishing groups of probes while still permitting individual identity to be resolved from other sequence regions (see Hacohen [0061], [0119]-[0120], and [0129] “Barcode sequences can be designed such that each sequence is correlated to a particular portion of nucleic acid, allowing sequence reads to be correlated back to the portion from which they came”). Directly reading on an index sequences that identifies the set of pools from which the concatemer originates. Hacohen further teaches multiple methods for applying a barcode/index to concatemer, including PCR addition to either end (see Hacohen [0129], and throughout).
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 3-6, 9-11, 13-20 are rejected under 35 U.S.C. 103 as being unpatentable over Hacohen et al. (US 2023/0235394 A1) as applied to claim 1, 3-5, 9-11, and 17-18 above, included here for reasons supra, and further in view of Mok et al. (US 2020/0010875 A1, published Jan. 9 , 2020, on IDS).
In regards to claim 6, Hacohen teaches all of the limitations of claim 1, included here for reasons supra, but does not expressly disclose a two-stage PCR architecture in which a first PCR generates a first PCR product and a distinct second PCR which modifies the product of the first PCR, in order to prepare the products for concatenation. Specifically, Hacohen does not teach a separate first PCR that generates an untailed PCR product. However, Mok discloses this exact two-round PCR architecture. Mok discloses that a reporter DNA molecule may be generated via a first PCR and then modified in a second PCR to add adapter sequences for concatemer assembly (see Mok Figs. 1, 4, 7-9 [0018]-[0020], [0089], and throughout). Such sequential PCR workflows are standard in molecular biology when preparing DNA fragments for ligation or sequencing.
It would have been obvious to one or ordinary skill in the art at the time of filing to apply Mok’s two-round adapter incorporation PCR scheme to prepare the pools used in Hacohen’s array-assembly method. One of ordinary skill would have been motivated to combine these teachings because Mok’s expressly explains the practical benefits of splitting the amplification into two rounds in this manner allows for simplifying primer design and synthesis when adapting the tailing scheme across a panel of many distinct target regions, a benefit equally applicable to Hacohen’s multi-family adapter design. One of ordinary skill would have had a reasonable expectation of success because both rounds are standard PCR amplifications using well understood principles and Mok’s own disclosed working examples confirm the two round scheme functions reliably to generate concatenated DNA for sequencing.
In regards to claims 13-16, Hacohen teaches the limitations of claim 1, included here for reasons supra. Hacohen doesn’t expressly disclose that the linear DNA concatemers are subjected to PCR to add at least a first sequencing adaptor to the concatemers, instead directing a person of ordinary skill in the art to “enter standard Nanopore or PacBio library prep workflows for subsequent sequencing” (see Hacohen [0095]) after forming the concatemers. Mok discloses specific steps for using PCR to add sequencing priming sites and adaptors on each end of the concatemers (see Mok [0042], [0122]-[0123], and throughout). A person of ordinary skill in the art would recognize that these sequencing preparation steps of adding adaptors or primer binding sites would be inter changeable and is common practice in the art.
In regards to claims 19 and 20, Mok teaches utilizing PCR to add sequencing adapters, sequencing primers and index sequences to the end of each concatemer (see Mok [0004], [0008]-[0012], [0015] [0017], [0032], [0042], [0047], and throughout). While Mok doesn’t explicitly state that the sequencing adaptor, sequencing primer binding site and an index sequence appear in that order, one of ordinary skill in the art would anticipate this to be the case for a sequencing assay. The sequencing adaptor must be located on the end in order for the entire concatemer to be sequenced. Furthermore the index must be located inside the amplification region in order for it to be useful as an index.
Claims 1, 3-22 are rejected under 35 U.S.C. 103 as being unpatentable over Hacohen (US 2023/0235394 A1) and Mok et al. (US 2020/0010875 A1, published Jan. 9, 2020, on IDS) as applied to claim 1, 3-6, 9-11, 13-20 above, included here for reasons supra, and further in view of Assarsson et al. (“Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability”, PLoS ONE 9(4), 2014, on IDS).
In regards to claims 7 and 8, Hacohen teaches all of the limitations of claim 5 as outlined above and included here for reasons supra, however Hacohen does not teach that the detection assay generating the input DNA is specifically a proximity extension assay (PEA) comprising an extension step and an amplification step.
As noted in the Applicant’s remarks, Assarsson teaches the use of PEA to generate reporter DNA fragments (see Abstract and throughout). Assarsson teaches and extension step (to generate the reporter DNA) and an amplification step occurring withing a single PCR reactions (see Assarsson pg. 3 right col. 2nd para.). This streamlined “single-tube” protocol is a core feature of PEA and would have been well understood and obvious to one of ordinary skill in the art.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use Assarsson’s PEA including its extension and amplification steps, as the detection method for generating input DNA for Hacohen method. Similar methods of using PEA to generate DNA used in sequencing readouts were already described in the art.1 Furthermore, one would have a reasonable expectation of success as Hacohen’s method is applicable to any DNA, no matter how it was originally generated.
Assarsson further discloses running multiple multiplex PEA panels on the same sample to assess scalability (see Assarsson Results “Demonstrating high specificity and scalability of PEA” disclosing “Scalability was further analyzed by measuring the level of 24 analytes in a healthy plasma sample either in 24-plex or in 96-plex PEA. This was done by generating two different probe mixes”). This discloses at least two panels of proximity probe pairs used on the same sample, reading on these limitations. The 24 plex panel and the 96-plex panel are each directed to a group of analytes and are therefore panels “for the detection of a different group of analytes” within the plain meaning of claim 8. Assarsson further discloses that within each panel every probe pair comprises a distinct pair of nucleic acid domains engineered to prevent non-matching probes from binding to each other. “a unique 5-base pair long annealing site that prevented non-matching probes to bind” was added “when increasing from 24- to 96 plex”. Confirming that each of the analyte-specific probe pairs used in a panel is designed with a distinct nucleotide domain pair, which may be reused across different panels (see Assarsson pg. 1 right col. 2nd para., Figs. 4, S1, Table S1, and throughout).
In regards to claim 12, Hacohen teaches forming linear concatemers using uracil specific excision reagent assembly comprising processing PCR products in each pool to generate 3’ overhangs comprising the assembly sites, combining the pools, and generating multiple linear DNA concatemers, with the PCR produces of each pool being joined to the PCR products of different pools having complimentary overhangs (see Hacohen Fig. 1,[0017], [0095]-[0097], [0102], and throughout). Hacohen further teaches sequencing the concatemers, thereby identifying the analytes detected in each (see Hacohen [0019], [0037]-[0038]). Hacohen doesn’t teach that the input DNA molecules are obtained from multiplex PEA. As discussed above in relation to claims 7 and 8, Assarsson teaches this limitation and it would be obvious to combine the teaching of Assarsson with Hacohen.
In regards to claim 21, the claim recites the consolidated limitations of many of the claims above and includes multiplexed proximity extension generated reporter DNA such at that taught by Assarsson as outlined in regards to claims 7 and 8, combined with the linear concatemer construction methods such as those taught by Hacohen as demonstrated in regards to claim 1. Hacohen teaches using PCR to generate DNA fragments compatible with USER® assembly and ordered concatenation which produce linear DNA fragment for sequencing. Mok likewise teaches concatenating short DNA fragments for use in sequencing reactions and teaches additional methods that are compatible with Hacohen’s methods.
Combined Hacohen, Mok, and Assarsson render obvious “A method of detecting multiple analytes in one or more samples” (see Hacohen (see Hacohen [0007], [0102]), “comprising: (i) performing multiple multiplex detection assays on one or more samples, in order to detect multiple analytes in each sample, wherein each multiplex detection assay is a proximity extension assay comprising an extension step that generates reporter DNA molecules” (see Assarsson pg. 1 right col. 2nd para., Figs. 4, S1, Table S1, and throughout), “and an amplification step in which the reporter DNA molecules are amplified, wherein the extension and amplification steps take place within a single PCR reaction and yield a pool of amplified reporter DNA molecules, each reporter DNA molecule being specific for an analyte” (see Assarsson pg. 3 right col. 2nd para.), “(ii) performing a PCR on each pool using assembly primers, wherein all the reporter DNA molecules in one pool are amplified using the same primer pair, and a different primer pair is used for amplification in each pool, and wherein each primer of the primer pairs comprises a unique assembly site which is complementary to one unique assembly site in one other pool” (see Hacohen [0017], [0102], Table 1); “(iii) combining the PCR products of each pool to form a combination pool (see Hacohen Fig. 1, [0095]-[0097]); (iv) in the combination pool, forming by uracil-specific excision reagent assembly linear DNA concatemers containing a PCR product of one reporter DNA molecule from each pool, wherein a position of each PCR product of a reporter DNA molecule within the concatemer correlates to the pool from which the reporter DNA molecule originated” (see Hacohen Figs. 6, 7, and 9, [0082]-[0085]); “(v) subjecting the concatemers to a single PCR reaction in which a sequencing adaptor, a sequencing primer binding site, and an index sequence are added to both ends of each concatemer” (see Mok [0004], [0008]-[0012], [0015] [0017], [0032], [0042], [0047], and throughout); “and (vi) sequencing the concatemers, thereby identifying the analytes detected in each proximity extension assay based on the combination of the sequence of each reporter DNA molecule and its position within its concatemer” (see Hacohen [0045], [0213]).
It would have been obvious to one ordinary skill in the art to combine the PEA generated reporter DNA with the concatenation and sequencing methods taught by Hacohen as similar methods of using PEA to generate DNA used in sequencing readouts were already described in the art.2 Further, Hacohen expressly touts a substantial increase in long-read sequencing throughput (see Hacohen [0093]), and a person of ordinary skill seeking to improve the throughput and scalability of Assarsson’s qPCR read proximity extension assay would have looked to Hacohen’s sequencing based approach as a known technique for achieving exactly that benefit (see MPEP 2143(I)(C)). Furthermore, one would have a reasonable expectation of success as Hacohen’s PCR based method for generating DNA for assembly is equally applicable to PEA generated reporter DNA, as the libraries of smaller fragments that Hacohen reports for starting material (see Hacohen [0006]).
In regards to claim 22, it would have been obvious to a person of ordinary skill I the art to assemble a kit combining Assarsson’s proximity-probe detection components, Hacohen’s assembly primers and USER®/Gibson assembly components, and Mok’s PCR primers configured to generate sequencing ready concatemers as described in claim 21 above. One or ordinary skill in the art would have been motivated to combine these teachings because a person seeking to commercially package and practice the combined teachings as outlined above would necessarily require a kit containing reagents for each step of that method, including a means of preparing the finished assembled construct for sequencing. One of ordinary skill in the art would have had a reasonable expectation of success because each kit component performs its established function independent of the other components presence in the kit and combining these known kit components would yield the predictable results of a complete reagent set for practicing the combined method of claim 21.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Regev (US 2019/0085324 A1, published Mar. 21, 2019) Evidence that combining PEA detection chemistry with sequencing based multiplex readouts was a recognized workable combination in the art independent of Assarsson’s own disclosure. (see [0429] “The UAI can be generated using methods described for the proximity ligation assay (PLA) or proximity extension assay (PEA)… PEA is based on pairs of antibodies that are linked to oligonucleotides having slight affinity to one another (PEA probes). Upon target binding the probes are brought in proximity, and the two oligonucleotides are extended by a DNA polymerase forming the UAI that now acts as a unique surrogate marker for the specific antigen”). Regev further cites Assarsson’s methods as examples for “isolating single cells into individual reaction chambers to perform PCR amplification or a proximity ligation/extension assay” (see [0456])
2 Regev (US 2019/0085324 A1, published Mar. 21, 2019)
Prosecution Insights