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
Claims 1-25 are pending.
Rejections not reiterated in this office action are withdrawn.
Priority
This application is a CON of 18/455,152 (08/24/2023)
18/455,152 is a CON of 18/188,504 (03/23/2023)
18/188,504 is a CON of 17/122,321 (12/15/2020, US 11634751)
17/122,321 is a CON of 14/941,433 (11/13/2015, US 10900065)
14/941,433 has PRO 62/080,055 (11/14/2014).
Applicant persuasively argued support for element “(h) a deoxyribonucleic acid (DNA) polymerase suitable for polymerase chain reaction (PCR) amplification” based on the priority document and what was known in the art at the time.
New Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-25 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor at the time the application was filed had possession of the claimed invention.
Claim 1, step (c)(i), was amended to include the following new language:
comprising complementarity to the whole transcriptome of the cell such that the plurality of nucleic acid molecules is collectively capable of annealing to the whole transcriptome of the cell
while Applicant stated the following regarding support for the amendment:
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The examiner reviewed the cited paragraphs and the entire disclosure and could not locate support for such a new limitation, nor does there appear to be a written description of the limitation in the application as filed. See Hyatt v. Dudas, 492 F.3d 1365, 1370, 83 USPQ2d 1373, 1376 (Fed. Cir. 2007) (holding that "[MPEP] § 2163.04 (I)(B) as written is a lawful formulation of the prima facie standard for a lack of written description rejection."). There is no literal support for the added limitation and one of skill in the art would not recognize that Applicant possessed the claim scope.
New 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.
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, 8-12, 19-23, 25 are rejected under 35 U.S.C. 103 as being unpatentable over Nolan et al. (WO2012106385) in view of Fan et al. (US20160258012, EFD 2013-08-28) and Salathia et al. (US20160053253, EFD 2014-04-29).
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 does not teach an embodiment of a “kit” or that a first hybridization sequence comprises complementarity to the whole transcriptome.
Fan teaches “nucleic acid analysis of single cells” (Title, Abstract) and determining the whole transcriptome of the cell (Claim 132: “amplifying the whole transcriptome of said one or more cells”; [0602]: “the entire transcriptome of each single cell in the sample may also be measured”) using techniques such as RNA-seq ([0613]: “single-cell transcriptional landscape by highly multiplex RNA-seq”; [0777]: “ the target binding region comprises a sequence is adapted to hybridize to a target nucleic acid. … the target nucleic acid comprises a plurality of target nucleic acids comprising at least … 100% of the transcripts of a transcriptome of an organism”).
Salathia teaches “single cell gene expression analysis” using “unique barcodes such as unique molecular barcodes (UMI)” (Title, Abstract, claim 1) and applying RNA-Seq for whole transcriptome analysis (claim 11; [0060]: “The methods presented herein can include methods of generating tagged cDNA with sample-specific tags”; [0061]: “The double stranded cDNA can then be converted into a sequencing library using for example NEXTERA™ or TRUSEQ™ (Illumina, Inc.) for whole transcriptome RNA-Seq”). Salathia teaches whole transcriptome sequencing using random hybridization primers ([0074]-[0081]).
One of ordinary skill in the art following the teaching of Nolan would have considered known techniques for single cell analysis as taught by Fan and Salathia including the use of RNA-Seq for whole transcriptome analysis via utilizing hybridization sequences that are complementary to the whole transcriptome as taught by Fan and Salathia. One of ordinary skill in the art would have also assembled all of the elements required to perform the modified technique of Nolan thereby preparing a “kit”. One of ordinary skill in the art would have had a reasonable expectation of success because each of Nolan, Fan, and Salathia are in the same field of endeavor of single cell transcriptome analysis and they would have been motivated by the specific teaching of Nolan to perform the technique in order to identify the target molecules in a cell using the known techniques suggested by Fan and Salathia. Thus, claim 1 is prima facie obvious.
Regarding claim 2, Nolan teaches that the 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”).
Regarding claim 3, 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 8, Nolan teaches t4 DNA ligase ([0084]: “ligation can be performed enzymatically by at least one DNA ligase or at least one RNA ligase, for example but not limited to, T4 DNA ligase”).
Regarding claims 9-11, Nolan teaches a capture region which used for isolation onto a bead ([0091]) including the use of biotin-streptavidin ([00102]) 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 12, Nolan teaches the tags are combined in a manner to uniquely identify the target molecules with a “split pool” approach ([00163]-[00170]) and 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”, “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”) where the annealing regions are different ([00111]: “The annealing regions incorporated to each end of an APS can be different.”) respectively corresponding to the claim’s 3rd, 4th, and 5th sequences.
Regarding claims 19-22, Nolan teaches utilizing wells and any suitable surface known in the art ([00164]: “Cell populations can be split into wells, bead or any suitable surfaces known in the art.”) and 96-well microtiter plates ([00208]: “e.g. 96 well or greater microtiter plates”; [00212]; [00221]; claim 309) which are routinely used in the art for preparation of such nucleic acid samples, including split-pool barcoding ([00163]-[00170]) such that one of ordinary skill in the art would consider their use including a distinct barcode for each well to efficiently implement the split-pool approach and arrive at the claimed invention.
Regarding claims 23, Nolan teaches a capture region which used for isolation onto a bead ([0091]) including the use of biotin-streptavidin ([00102]) 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 25, Nolan teaches greater than 1 million cells ([0018]-[0020]: “x is greater than 1,000,000”, x is number of cells).
With each of the above 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. Thus, one of ordinary skill in the art would have arrived at the invention as claimed with a reasonable expectation of success before the effective filing date of the claimed invention.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Nolan et al. (WO2012106385) in view of Fan et al. (US20160258012, EFD 2013-08-28) and Salathia et al. (US20160053253, EFD 2014-04-29) as applied to claims 1-3, 8-12, 19-23, 25 above and further in view of Nolan et al. (US20140099637)(“Nolan-2014”).
Regarding claim 4, Nolan teaches fixation but does not teach use of formaldehyde ([00300]: “The cells are immediately fixed and permeabilized by adding 50 mL cold methanol.”).
Nolan-2014 teaches use of common fixing agents including formaldehyde in a related technique of detecting nucleic acids from a cell ([0050]; Abstract; claim 1) that one of ordinary skill in the art would have considered in combination with Nolan to allow preservation of the cell.
Claims 5-7, 13-14, 24 are rejected under 35 U.S.C. 103 as being unpatentable over Nolan et al. (WO2012106385) in view of Fan et al. (US20160258012, EFD 2013-08-28) and Salathia et al. (US20160053253, EFD 2014-04-29) as applied to claims 1-3, 8-12, 19-23, 25 above and further in view of Johnson et al. (US20140057799).
Regarding claims 5-6, Nolan teaches the cell is lysed but does not teach a specific reagent, however, Johnson teaches a related technique for studying nucleic acids in a fixed, permeabilized, single cells and performs lysis using proteinase K which is well-known and routinely used in the art ([0168]).
Regarding claim 7, Nolan teaches reverse transcription ([0059]) but not the particular enzyme reverse transcriptase, however, Johnson teaches use of reverse transcriptase ([0078]) well-known and routinely used in the art.
Regarding claims 13-14, Johnson teaches forward and reverse primers ([0014]) which comprise barcodes ([0082]; [0155]) while Nolan teaches sequencing the product using NGS systems ([00133]-[00142]) and including through the use of sequencing primers ([00270]; [00277]: “comprises a second amplification primer complimentary region for hybridization of PCR or sequencing primers.”; [00283]), such that one of ordinary skill in the art would have incorporated NGS sequencing primer sequence in the amplification primers in the course of preparing for sequencing and arrive at the claimed invention with a reasonable expectation of success.
Regarding claim 24, Nolan teaches the use of beads as per instant claim 23, but does not specifically teach SPRI beads. Johnson teaches use of SPRI beads of the same type as disclosed in the instant specification ([0147]: “Ampure bead technology (PerkinElmer)”) which one of ordinary skill in the art routinely uses in oligonucleotide purification.
Claims 15-18 are rejected under 35 U.S.C. 103 as being unpatentable over Nolan et al. (WO2012106385) in view of Fan et al. (US20160258012, EFD 2013-08-28) and Salathia et al. (US20160053253, EFD 2014-04-29) as applied to claims 1-3, 8-12, 19-23, 25 above and further in view of Islam (“From Single-Cell Transcriptomics to Single-Molecule Counting”, Thesis - Karolinska Institutet (Sweden). 2013. 59 pages).
Regarding claims 15-18, Nolan teaches using reverse transcription in preparation for sequencing ([0063], [0065]) but does not specifically teach the use of a poly(T), random hexamer, or transcript-specific sequences. However, Islam teaches that the use of these elements are part of standard sequencing library preparation in in reverse transcription (p. 15: “In standard library preparation for RNA-seq experiments, the RT reaction is primed by different types of primer and different RNA priming strategy can effect the efficiency of cDNA synthesis. Three priming strategies are available: sequence-specific primer, oligo (dT) primer and random hexamer.”) such that one of ordinary skill in the art would have reasonably considered their use and arrive at the claimed invention.
Conclusion
No claims allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ROBERT H HAVLIN whose telephone number is (571)272-9066. The examiner can normally be reached 9am - 6pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Kortney Klinkel can be reached at (571) 270-5293. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ROBERT H HAVLIN/Primary Patent Examiner, Art Unit 1626