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 and Formal matters
This action is in response to papers filed 11/26/2025.
Claim 1 has been amended.
Claims 28-29 has been added by amendment.
Applicant’s election without traverse of group I, 1-23, 1) an oligonucleotide barcode of the plurality of oligonucleotide barcode, (i) the complement of the target-binding region comprises the reverse complementary sequence of the target-binding region and/or the complementary sequence of the target-binding region, “wherein hybridizing the complement of the target-binding region of a barcoded nucleic acid molecule with the target-binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises intermolecular hybridization of the complement of the target-binding region of a barcoded nucleic acid molecule with the target-binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes, wherein the second molecular label is a different from the first molecular label, and wherein the second molecular label is not a complement of the first molecular label” recited in claim 12, (i) the complement of the target-binding region comprises the reverse complementary sequence of the target-binding region and/or the complementary sequence of the target-binding region, wherein the one or more blocker oligonucleotides are capable of specifically binding to a portion of the first universal sequence, wherein the one or more blocker oligonucleotides (vii) comprise a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof. in the reply filed on 10/10/2022 is acknowledged.
Claims 11, 13, 24-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention/species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/10/2022.
Claims 1-10, 12, 14-20, 23, 28-29, are being examined.
Any rejection or objection not reproduced below have been withdrawn in view of amendments.
Priority
The application was filed 01/29/2021 and claims priority from provisional application 62968947, filed 01/31/2020.
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 28-29 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 28 has been amended to recite, “wherein the portion of the oligonucleotide barcode comprises the first universal sequence, the cell label, the molecular label, the target-binding region, portions thereof, or any combination thereof..” The recitation of portions thereof is confusing and unclear.
Claim 28 has been added by amendment to recite, “The cell label.” The claim depends from .claim 1 which does not provide antecedent basis. Thus the metes and bounds are unclear what the limitation is referencing.
Response to Arguments
This is a new grounds or rejection necessitated by amendment.
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 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.
Claim(s) 1-10, 12, 14-20, 23, 28-29 is/are rejected under 35 U.S.C. 103 as being unpatentable Mikkelsen (WO2018075693),), Vestheim (Methods in Molecular Biology (2011) pages 265-273) .
The specification teaches, “A universal sequence can be a region of nucleotide sequence that is common to two or more nucleic acid molecules.” (0057) Thus a universal sequence could broadly be encompassed any 2 nucleotides that are present in two or more nucleic acid molecules.
The specification teaches, “The associated nucleic acids can comprise one or more of spatial labels, target labels, sample labels, indexing label, or barcode sequences (e.g., molecular labels) (0057). The specification teaches, “0107] A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length.” Thus a molecular label encompasses about a single nucleotide. It could also encompass any type of label, in view of the lack of a limiting definition in the specification.
Further the claim requires a target binding region of the oligonucleotide barcodes which comprises, “an oligo dT” and is capable of hybridizing to the nucleic acid target under any conditions. Thus the target binding region can be two deoxy thymine nucleotides.
Thus the oligonucleotide comprises a universal sequence (at least 2 nucleotides), a molecular label (about a nucleotide or other label) and at least two thymines.
Mikkelsen teaches, “[00284] The present disclosure also provides methods for reducing nonspecific priming in a single-cell 5' gene expression assay. In generating an assay that allows measurement of 1) a cell barcode sequence (barcode), 2) a unique molecular identifier sequence (UMI) and 3) the 5' sequence of an mRNA transcript simultaneously, one strategy is to place these sequences on a sequence that attaches to the 5' end of an mRNA transcript -- in the present disclosure, this may be accomplished by placing the barcode and UMI on a template switching oligonucleotide (TSO). This oligonucleotide may be attached to the first strand cDNA via a template switching reaction where the reverse transcription (RT) enzyme 1) reverse transcribes a messenger RNA (mRNA) sequence into first-strand complementary DNA (cDNA) from a primer targeting the 3' end of the mRNA, 2) adds nontemplated cytidines to the 5' end of the first-strand cDNA, 3) switches template to the TSO, which may contain 3' guanidines or guanidine-derivatives that hybridize to the added cytidines. The result is a first-strand cDNA molecule that is complementary to the TSO sequence: cell-barcode, UMI, guanidines, and the 5' end of the mRNA. [00285] In some cases, the TSO may co-exist in solution with the RT enzyme and the total RNA contents of a cell. If the TSO is a single stranded DNA (ssDNA) molecule, it can participate as an RT primer rather than as a template-switching substrate. Given, for example, that the over 90% of the total RNA contents of a cell include noncoding ribosomal RNA (rRNA), this may produce barcoded off products that do not contribute to the 5' gene expression or V(D)J sequencing assay but do consume sequencing reads, increasing the cost required to achieve the same sequencing depth. In addition, if the UMI is implemented as a randomer, the presence of this randomer at the 3' end of the TSO greatly increases its ability to serve as a primer on rRNA template. [00286] In some cases, a TSO that is less likely to serve as an RT primer via the introduction of a particular spacer sequence between the UMI and terminal riboGs may be used. Another approach is to design and include a set of auxiliary blocking oligonucleotides that may hybridize to rRNA and prevent binding of the TSO. [00287] The spacer sequence can be optimized by selecting a sequence that minimizes the predicted melting temperature of the (spacer-GGG):rRNA duplex against all human ribosomal RNA molecules. [00288] The blocker sequences can be optimized by selecting sequences that maximize the predicted melting temperature of the (blocker):rRNA duplex against all human ribosomal RNA molecules.
Mikkelsen teaches methods of labeling nucleic acids according to the methods of claim 1 including the use of blocking oligonucleotides (00284-00291).
Mikkelsen does not specifically teach blocking oligonucleotides to reduce generation of first universal sequence.
However, Vestheim teaches, “ “Universal” or group-specific PCR primers have a tendency to predominately hybridize with the common sequences in samples with mixed templates. The result is that the rarer sequences are seldom retrieved by cloning or sequencing. The use of a blocking oligonucleotide (oligo) designed to specifically prevent amplification of dominant or unwanted DNA templates is an easy way to improve the amplification of rarer sequences. Here, we describe the different types of blocking principles and the different types of blocking oligos and give guidelines and examples of their application.” (abstract). Vestheim continues by providing methods and protocols for the design of blocking oligonucleotides.
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to specifically design and use blocking oligonucleotides to inhibit extension of universal sequences in the method of Mikkelsen. The artisan would be motivated as Vestheim teaches blocking allows for retrieval of rarer sequences. The artisan would have a reasonable expectation of success as the artisan is merely specific guidance of Vestheim in the method of Mikkelsen.
Mickelsen teaches, “In some embodiments, the initial poly-T primer, comprising sequences 2016 and 2014 can be attached to a gel bead as opposed to the TSO.” (302)
With regards to claim 2-3, Mikkelsen teaches determining number of sequencing reads (00297).
With regards to claims 4, Mikkelsen teaches target switching oligonucleotides which have labels and are intermolecular (00284-00297).
With regards to claim 5, Mikkelsen teaches denaturing prior to hybridization (00327).
With regards to claim 6, Mikkelsen teaches amplification of labeled nucleic acids. (00327).
With regards to claim 7, Mikkelsen teaches the TSO targets the 3’ of mRNA. Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims the teachings of target the 3’ end of the mRNA is having a portion complementary to the 3’ end of the mRNA. The artisan would be motivated to provide a sequence complementary to the 3’ end of the mRNA to target the mRNA. The artisan would have a reasonable expectation of success as this is an art accepted method of targeting a nucleic acid. (00284).
With regards to claim 8, Mikkelsen teaches a UMI and a cell label. Mikkelsen does not teach they are complementary. (00284).
With regards to claim 9, 10, 12, 16, 20 Mikkelsen teaches a UMI, a cell label, and mRNA. (00284).
With regards to claim 15, Mikkelsen teaches the template switching oligonucleotide (TSO) has riboG (00300).
With regards to claim 17-18, Vestheim teaches “ a moderately effective blocking oligo (e.g. producing a 70% reduction in amplification efficiency of non-target template” (266, 1st full paragraph).
With regards to claim 19, Mikkelsen teaches the use of sequencing adapter (00330).
With regards to claim 23, Vestheim teaches oligonucleotides with PNA, LNA of 25 nucleotides and a Tm slightly higher than then primers. Mikkelsen teaches blocker sequences with Tm higher than 45oC. (table 2). Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims the art of Mikkelsen and Vestheim render obvious (i) is 1 nt to 100 nt long; (ii) have a Tm of at least 45°C; (iii) have a Tm of at most 45°C; (iv) do not comprise non-natural nucleotides; (v) are unable to function as a primer for a reverse transcriptase or a polymerase; (vi) comprise non-natural nucleotides; and/or (vii) comprise a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof. The artisan would be motivated to block reverse transcription or amplification of universal sequence to allow detection of rare sequences. The artisan would have a reasonable expectation of success as the artisan is merely applying the art.
With regards to claim 28, Mikklsen teaches, “0037] In some embodiments, the first defined sequence comprises at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site. In some embodiments, the second defined sequence comprises at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site.”
With regards to claim 29, Vestheim teaches, “A blocking oligo can be designed to overlap with one of the uni versal primers (called an annealing inhibiting blocking oligo because when bound to the target, it prevents annealing of the universal primer) (Fig. 2a). It can also be positioned between the two universal primers, called an elongation arrest blocking oligo (Fig. 2b). Design an annealing inhibiting blocking oligo if possi ble as these have proved to be more efficient than elongation arrest blockers (4, 11, 12)” Thus Vestheim teaches blockers are complementary to a universal sequence.
Response to Arguments
The response begins traversing the rejection by reproducing portions of the rejection..
The rejection traverses the rejection asserting the art does not teach extending and using a template switching oligonucleotide. This argument has been thoroughly reviewed but is not considered persuasive as Mikklsen teaches, ““[00284] The present disclosure also provides methods for reducing nonspecific priming in a single-cell 5' gene expression assay. In generating an assay that allows measurement of 1) a cell barcode sequence (barcode), 2) a unique molecular identifier sequence (UMI) and 3) the 5' sequence of an mRNA transcript simultaneously, one strategy is to place these sequences on a sequence that attaches to the 5' end of an mRNA transcript -- in the present disclosure, this may be accomplished by placing the barcode and UMI on a template switching oligonucleotide (TSO). This oligonucleotide may be attached to the first strand cDNA via a template switching reaction where the reverse transcription (RT) enzyme 1) reverse transcribes a messenger RNA (mRNA) sequence into first-strand complementary DNA (cDNA) from a primer targeting the 3' end of the mRNA, 2) adds nontemplated cytidines to the 5' end of the first-strand cDNA, 3) switches template to the TSO, which may contain 3' guanidines or guanidine-derivatives that hybridize to the added cytidines. The result is a first-strand cDNA molecule that is complementary to the TSO sequence: cell-barcode, UMI, guanidines, and the 5' end of the mRNA. [00285] In some cases, the TSO may co-exist in solution with the RT enzyme and the total RNA contents of a cell. If the TSO is a single stranded DNA (ssDNA) molecule, it can participate as an RT primer rather than as a template-switching substrate. Given, for example, that the over 90% of the total RNA contents of a cell include noncoding ribosomal RNA (rRNA), this may produce barcoded off products that do not contribute to the 5' gene expression or V(D)J sequencing assay but do consume sequencing reads, increasing the cost required to achieve the same sequencing depth. In addition, if the UMI is implemented as a randomer, the presence of this randomer at the 3' end of the TSO greatly increases its ability to serve as a primer on rRNA template. “ Thus Mikkelsen teaches RT of barcoded oligonucleotides using TSO (template switching oligonucleotide).
The response continues by providing arguments with respect to hybridizing of the complement of the target binding region to target binding region of a plurality of oligonucleotides. This argument has been thoroughly reviewed but is not considered persuasive as the RT provides for this hybridization.
The response further provides arguments with respect to elongation. These arguments are not persuasive in view to the teachings of 0284-0285.
Summary
No claims are allowed.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Sham (US20180216174)
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 STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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