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 .
Status
Claim rejections and objections not reiterated in this action are withdrawn.
Claims 1-22 are pending.
Priority
This application is a CON of 17/122,321 (12/15/2020, now US11634751),
17/122,321 is a CON of 14/941,433 (11/13/2015, now US10900065),
14/941,433 has PRO 62/080,055 (11/14/2014).
New Claim Rejections - 35 USC § 103
Claims 1-22 are rejected under 35 U.S.C. 103 as being unpatentable over Ke et al. (Nat Methods 10, 2013-07-14, p. 857–860 and Supplemental, 35 pages) in view of Saliba et al. (Nucleic Acids Research, 2014-07-22, Vol. 42, No. 14, p. 8845–8860) and Nolan et al. (WO2012106385).
Ke teaches in situ sequencing in cells as depicted in Fig. 1a:
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through providing fixed and permeabilized cells (p. 5: “Fixation was performed in 3% (w/v) paraformaldehyde”, “The tissue was permeabilized in 2 mg/ml pepsin”), generating cDNA via RT using primers (p. 5: “In situ reverse transcription”, “cDNA primer or 5 μM of unmodified random decamers (all oligonucleotides sequences are listed in Supplementary Tables 4–6)”), tagging with barcode sequence (p. 5: “Barcode padlock probing”).
Regarding claim 1, Ke teaches the steps of the claims including tagging with a barcode but not the specific split-pool barcoding steps of dividing, coupling, and combining.
Nolan teaches tagging target molecules of nucleic acids, including RNA, from transcripts in a cell (claims 1-23; [0059]; [0069]: “individually tagging cells”; [00250]-[00256]; claims 69-112; [00152]; [0063]; [00192]: “target nucleic acids may be obtained from a single cell.”; [00195]: “detect a novel transcript in order to diagnose or condition”; claims 261-312). Nolan teaches fixing the cells ([0006]: “In some embodiments, the invention relates to methods for identifying whether a plurality of targets are in a plurality of cells comprising: binding to the targets a plurality of tags, wherein a tag comprises a code that represents a) the target identity and b) the identity of the cell in which tag is binding. ... In some embodiments, the cell is lysed or fixed.”)
Nolan teaches permeabilizing the cells ([00116]: “Cells may be fixed prior to the addition of UBAs, ESBs or prior to COB assembly. Suitable cell permeabilization methods are known in the art and can be used to deliver components of the assay into cells and cellular components.”).
Nolan teaches generating cDNA by reverse transcription ([0059], [0063]), but not the use of the RT primers as claimed. Nolan teaches tagging in a manner to uniquely identify the target molecules with a “split pool” approach including multiple rounds where the number of unique tags are numerically governed by the length (claims 196-197, [0010], [00110]-[00111], [00163]-[00170]; Fig. 4).
Nolan is in the same field of endeavor of single cell analysis with details regarding unique barcoding including split-pool barcoding ([00164]; Fig 4). Nolan teaches kits for tagging target molecules of nucleic acids from transcripts ([0069]: “kits for individually tagging cells”; [00250]-[00256]; claims 69-112; [00152]; [0063]; [00192]: “target nucleic acids may be obtained from a single cell.”; [00195]: “detect a novel transcript in order to diagnose or condition”). Nolan teaches fixing the cells ([0006]: “In some embodiments, the invention relates to methods for identifying whether a plurality of targets are in a plurality of cells comprising: binding to the targets a plurality of tags, wherein a tag comprises a code that represents a) the target identity and b) the identity of the cell in which tag is binding. ... In some embodiments, the cell is lysed or fixed.”). Nolan teaches permeabilizing the cells ([00116]: “Cells may be fixed prior to the addition of UBAs, ESBs or prior to COB assembly. Suitable cell permeabilization methods are known in the art and can be used to deliver components of the assay into cells and cellular components.”). Nolan teaches single-stranded tags / UBAs ([0010]: “In some embodiments, the tag comprises a UBA.”; [0082]: “In some embodiments, the UBA is an aptamer. Aptamers include nucleic acid aptamers (i.e., single-stranded DNA molecules …”) which are configured to hybridize to the transcriptome of the cell ([00229]: “the UBAs, e.g., oligonucleotide probe, have substantially the same length so that they hybridize to target nucleotide sequences at substantially similar hybridization conditions. As a result, the process of the present invention is able to detect infectious diseases, genetic diseases, and cancer”; [0064]; [00192]: “target nucleic acids may be obtained from a single cell.”; [00195]: “detect a novel transcript in order to diagnose or condition”) and has a second sequence that is the same in each of the nucleic acid molecules ([00110]: “The COB can be attached to the UBA via a common linker (CL). The CL can also be part of an oligonucleotide”). Nolan teaches a set of tags comprising multiple distinct barcode sequences comprising a sequence complementary to a common sequence ([00110]: “Substantially complementary or exact complementary annealing regions may be utilized for hybridization. An annealing region may be provided on both ends of an oligonucleotide ESB or APS. In some embodiments, the APSs are added in various steps of a split pool synthesis or any other suitable stepwise synthesis known in the art. An annealing region specific to each step of a stepwise synthesis maybe incorporated into the oligonucleotides”; [00111]-[00113]: “APSs can be designed to hybridize to the CL”). Nolan teaches ligation ([0021]: “the method further comprises ligation”; [0084]; [00121]; [00170]). Nolan teaches lysing the cell ([0006]: “In some embodiments, the cell is lysed”). Nolan teaches PCR amplification of DNA using polymerase ([0063]: “PCR amplification”; [00115]; [00125]: “the assembled products are amplified”, “products are amplified by polymerase chain reaction (PCR).”; [00191]; [00273]: “The COBs are optionally PCR amplified for sequencing using primers targeting the amplification primer complementary regions on the CL and the last APS subunit.”; [00170]: “extended via polymerases”; [0059]).
Nolan teaches the tags are combined in a manner to uniquely identify the target molecules with a “split pool” approach where the number of unique tags are generated by dividing, coupling an aliquot-specific barcode, and pooling (Fig. 4; [00163]-[00170]). One of ordinary skill in the art would have had a reasonable expectation of success in utilizing Nolan’s split-pool barcoding technique combined with the method of Ke because Nolan teaches it in the same context (cellular barcoding) and it was well-known in the art.
Saliba reviews single-cell RNA-seq (Title, Abstract) including utilizing the cell as a compartment to perform RNA-seq (p. 8851: “Sub-single-cell sequencing: localizing transcripts within a cell”, “A first strategy consists of isolating a compartment of a cell and applying the previously described RNA-seq techniques”). Saliba also describes details of the RNA-seq technique in Fig. 3 (p. 8851-52: “massively parallel RNA-seq of single cells (Figure 3)”) which when not performed within a cell includes lysis and isolation (Fig. 3: “Cell lysis” “bead immobilization”).
One of ordinary skill in the art following the teaching of Ke would have considered the teaching of Saliba (which cites to Ke) and Nolan which are in the same field of endeavor of single cell sequence analysis. One of ordinary skill in the art would have considered combining the known technique of RNA-seq adapted for an in situ manner as taught by Ke and modify the steps to perform lysis and isolation after RT and tagging to maintain the integrity of the cell for use as a compartment as specifically suggested by Saliba. One of ordinary skill in the art would have also utilized the well-known split-pool technique taught by Nolan to generate unique barcodes for identification. The level of skill in the art is very high as evidenced by the cited references and one of ordinary skill in the art routinely modifies and adapts known techniques to improve the information that can be derived from samples. Thus claim 1 is prima facie obvious.
Regarding claims 2 and 3 further dividing, lysing, and amplifying the isolated cDNA for sequencing, Ke teaches amplification and sequencing (p. 1, Fig. 1) as does Saliba (p. 8851, Fig. 3: “library preparation”, “library sequencing”, “Cell lysis”). Similarly, Nolan teaches detecting the nucleic acids via sequencing and lysing the cell ([0009]; [0021]-[0025]; claims 261, 278; [0006]: “In some embodiments, the cell is lysed”). Nolan teaches PCR amplification of DNA using polymerase ([0063]: “PCR amplification”; [00115]; [00125]: “the assembled products are amplified”, “products are amplified by polymerase chain reaction (PCR).”; [00191]; [00273]: “The COBs are optionally PCR amplified for sequencing using primers targeting the amplification primer complementary regions on the CL and the last APS subunit.”; [00170]: “extended via polymerases”; [0059]). One of ordinary skill in the art would consider applying the same steps as to obtain sequence information from the cell in the same manner as in the prior art.
Regarding claim 4-5, Nolan teaches the tags are combined in a manner to uniquely identify the target molecules with a “split pool” approach where the number of unique tags are numerically governed by the length (claim 264; [00163]-[00170]). Thus, one of ordinary skill in the art would have considered optimizing the length of the barcode tag oligonucleotides in order to provide a sufficient number of unique tags and arrive at the claimed invention.
Regarding claim 6, Nolan teaches nucleic acid tags comprising ends flanking the barcode sequence (Figs. 2, 4 – APS-CL; [0018]; [00164]).
Regarding claim 7-9, Nolan teaches a set of tags comprising multiple distinct barcode sequences comprising a sequence complementary to a common sequence linked by ligation including an 5’ overhang from a previously coupled APS-CL ([0010]; [00110]: “Substantially complementary or exact complementary annealing regions may be utilized for hybridization. An annealing region may be provided on both ends of an oligonucleotide ESB or APS. In some embodiments, the APSs are added in various steps of a split pool synthesis or any other suitable stepwise synthesis known in the art. An annealing region specific to each step of a stepwise synthesis maybe incorporated into the oligonucleotides”; [00111]-[00113]: “APSs can be designed to hybridize to the CL”; [0021]: “the method further comprises ligation”; [0084]; [00121]; [00170]; Figs. 2, 4 – APS-CL; [0018]; [00164]).
Regarding claim 10, Nolan teaches an equivalent of stop oligos ([00271]).
Regarding claim 11, Nolan teaches the nucleic acid tags are DNA ([0010]; [0082]; [00131]: “The APS subunits or entire COBs can be detected via full sequencing of all DNA tags by any suitable methods known in the art, e.g., Illumina HiSeq 2000, including the sequencing methods described herein”; [00171]).
Regarding claims 12-13, Nolan teaches a capture region which used for isolation onto a bead ([0091]) including the use of biotin-streptavidin ([00102]) as does Saliba (Fig 3) which were well-known and routinely used such that one of ordinary skill in the art would readily consider use for isolation and arrive at the claimed invention.
Regarding claim 14, Nolan teaches the tags are combined in a manner to uniquely identify the target molecules with a “split pool” approach where the number of unique tags are numerically governed by the length ([00163]-[00170]). Thus, one of ordinary skill in the art would have considered optimizing the length of the barcode tag oligonucleotides among the typical lengths ([0086]: “The nucleic acid sequence is preferably at least 15 nucleotides in length, and more preferably is at least 20 nucleotides in length.”) in order to provide a sufficient number of unique tags including 8 or more nucleotides and arrive at the claimed invention.
Regarding claim 15, Nolan teaches the biomolecular samples include cells derived from mammals ([00185]).
Regarding claims 16-17, Nolan and Saliba (Fig 3) teach RT primers of poly-T and Ke and Nolan teach random hexamers.
Regarding claim 18, Nolan teaches sequencing of the amplified DNA molecules as with claim 2.
Regarding claim 19-21, Nolan teaches analysis by sequencing the barcode/APS/COD to uniquely identify the cells by their tags ([00128]. [00131]-[00142], [00169]-[00181]) which one of ordinary skill in the art would consider grouping the reads according to the barcode and associated index sequences and arrive at the claimed invention.
Regarding claim 22, Nolan teaches the use of a 96-well plate ([00208]: “e.g. 96 well or greater microtiter plates”; [00212]; [00221]; claim 309).
With each of the claims, the level of skill in the art is very high such that one of ordinary skill in the art would consider routine the combination of elements from the teaching of the art in the same field of endeavor. One of ordinary skill in the art would have recognized that the results of the combination would be predictable due to the well-known nature and optimizations routinely performed in the art. Thus, one of ordinary skill in the art would have arrived at the invention as claimed with a reasonable expectation of success.
Therefore, the claims are rejected as prima facie obvious.
Conclusion
No claims allowed.
Applicant's submission of an information disclosure statement under 37 CFR 1.97(c) with the timing fee set forth in 37 CFR 1.17(p) on 1/12/26 prompted the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 609.04(b). 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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/ROBERT H HAVLIN/Primary Patent Examiner, Art Unit 1626
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