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 .
Status of claims / Response to Amendment
This office action is in response to an amendment filed on March 20, 2026.
Claims 1, 4-6 and 8-9 were previously pending. Applicant Amended claims 1 and 8; cancelled claim 4; and added new claims 16-17.
Claims 1, 5-6, 8-9 and 16-17 are currently pending and under consideration.
Applicant's claim amendments overcame the following objection and rejections:
objection to claim 1;
Rejections of Claims 1, 4-6 and 9 under 35 U.S.C. 112(b), in claims 1 and 4;
Rejection of Claim 8 under 35 U.S.C. 112(d);
Rejections of Claims 1, 4-6 and 9 under 35 U.S.C. 112(a) in claim 1;
Rejections of Claims 1, 4-6 and 8-9 under 35 U.S.C. 103 as being unpatentable over Larson.
The previously set forth 103 rejections have been withdrawn in view of the recent claim amendment filed on March 20, 2026, which added new limitations to the claims (i.e., the newly amended method in claim 1 requires performing each step "in the single reaction volume") and removed some of the previously considered features (i.e., the limitation "wherein the barcode is not biotinylated" has been removed). Thus, the scope of the claims has been changed in a manner that were not considered in the previous rejections.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
This office action contains new grounds of prior art rejections necessitated by Applicant's amendments. Although the claims were previously rejected as being unpatentable over the same reference(s), Applicant's amendments have necessitated the inclusion of new grounds of rejections in this Office action.
It is noted that, to the extent that they apply to the present rejection; Applicant's arguments are addressed below.
Priority -- Updated
For the instant claims 1, 5-6, 8-9 and 16-17 in this U.S. Application, the applicant claims priority of US provisional Application NO. 62/888,963, which has a filling date on 08/19/2019.
Response to Arguments
Applicant's arguments filed on March 20, 2026 have been fully considered.
Claim Rejections - 35 USC § 103
The previously set forth 103 rejections of claims 1, 4-6 and 8-9 have been withdrawn in view of the recent claim amendment filed on March 20, 2026, which included a new combination of limitations to the claims, that were not addressed in the previous rejections.
Applicant's arguments filed on March 20, 2026 have been fully considered but are not found persuasive.
Applicant argues that, because Larson teaches separation of double-stranded cDNA and cfDNA after second-strand synthesis, it does not teach or suggest the claimed method as amended, reciting a "method of simultaneously preparing a mixture of RNA and DNA targets in a single reaction volume for sequencing." (Remarks, pages 11-16)
This argument is not persuasive because it is not commensurate with the scope of the claim. As written, the claimed method does not exclude prior art methods that process a mixture of RNA and DNA in a single reaction volume, and subsequently separate the resulting products ꟷ "double-stranded DNA and double-stranded cDNA" ꟷ after a mixture comprising the products has been formed (as required by part (d) in claim 1). The preamble merely recites "simultaneously preparing a mixture of RNA and DNA targets in a single reaction volume," which describes the starting materials, but does not impose any limitation on how the resulting mixture is subsequently processed. Rather, the claimed method ends with forming a mixture of double-stranded cDNA and DNA.
Neither the claim nor the specification expressly excludes separation of double-stranded cDNA and DNA after second-strand synthesis. The disclosure does not describe maintaining all end-to-end library preparation steps in a single tube as a critical feature of the disclosed invention. In fact, the specification explicitly states that, after generating the double-stranded cDNA and DNA mixture, the user is free to process them for sequencing in a non-restrictive manner:
"[0036] Referring to FIG. 3, after step D, the sample now contains fully double stranded and A-tailed cDNA 300 and fully double stranded and A-tailed DNA 301. Notably, DNA 300 is distinguishable from DNA 301 by the presence of barcode 302 identifying the progeny of RNA from the progeny of DNA. The cDNA 300 and DNA 301 are ready for further steps in the sequencing workflow such as, for example adaptor ligation, amplification or target capture or any combination of the foregoing in any order desired by the user." [emphasis added]
Accordingly, although Larson teaches separation of double-stranded cDNA and cfDNA via biotin capture, it nonetheless anticipates the method of claim 1.
Claim Interpretation -- Updated
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, claim 1 has been amended to recite:
A method of simultaneously preparing a mixture of RNA and DNA targets in a single reaction volume for sequencing, wherein the method comprises the following steps:
(a) providing a sample in the single reaction volume,
wherein the sample comprises both RNA and DNA targets;
(b) contacting the sample with a target-specific primer in the single vessel, under conditions that do not allow DNA denaturation,
wherein the target-specific primer hybridizes to at least one RNA target,
wherein the target-specific primer comprises a nucleic acid barcode,
wherein the nucleic acid barcode distinguishes cDNA molecules from DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA;
(c) extending the target-specific primer hybridized to the at least one RNA target with a nucleic acid polymerase in the single reaction volume,
wherein the nucleic acid polymerase has reverse transcriptase activity, thereby forming a cDNA strand,
wherein the nucleic acid barcode is added to the cDNA strand by the reverse transcriptase activity; and
(d) contacting the sample with: (i) an RNaseH, and (ii) at least one nucleic acid end repair enzyme, in the single reaction volume, thereby forming a mixture of double-stranded DNA and double-stranded cDNA.
Thus, claim 1 recites method steps for processing RNA into barcoded double-stranded cDNA, while no specific processing is performed on the "DNA targets" that are also present in the sample mixture. Although the claim recites "DNA targets," it does not specify any structure limitation for this term, or what the DNA is target for. Thus, the term "DNA targets" broadly encompasses any DNA molecules, as any DNA molecules can serve as targets for a variety of activities such as depletion, capture, isolation, ligation, amplification, or other processes.
Therefore, any prior art method that teaches the steps of processing RNA as described, while having DNA present in the same reaction volume (including background DNA), would fall within the scope of the claimed method.
For the purpose of applying prior art, claim 1 recites "target-specific primer," which is not defined in the applicant's disclosure.
The specification provides the following relevant description regarding "target specific primer": "In some embodiments, the invention utilizes target-specific primers. A target specific primer comprises at least a portion that is complementary to the target." ([0039])
Thus, the term "target-specific primer" is interpreted under BRI and in light of the specification as encompassing primer comprising at least a portion of sequence that is complementary to another nucleic acid sequence (e.g. target nucleic acid). Therefore, random primers are encompassed by this term, as they hybridize to various regions on the target nucleic acid through complementary base-pairing.
Claim 1 recites the term "nucleic acid barcode," which is defined by the applicant's disclosure: "[t]he term "barcode" refers to a nucleic acid sequence that can be detected and identified." (para. [0014])
New Grounds of 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.
Claims 1, 5-6, 8-9 and 16-17 are rejected under 35 U.S.C. 112(b), 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.
Regarding claim 1, it has been amended to recite "(b) contacting the sample with a target-specific primer in the single vessel," which is indefinite because "the single vessel" lacks antecedent basis. The claim does not previously introduce a "single vessel," but instead recites "a single reaction volume." It is unclear whether "the single vessel" refers to "the single reaction volume" or something else entirely.
For the purpose of compact prosecution and applying prior art under 35 USC§ 102 and 103, "the single vessel" is construed as the container holding the "single reaction volume."
Claims 5-6, 8-9 and 16-17 are rejected for depending from claim 1 and not remedying the indefiniteness.
New Grounds of 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 following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 5-6, 8-9 and 16-17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Larson (US20180002749A1 - Differential tagging of rna for preparation of a cell-free dna/rna sequencing library; Published on 2018-01-04), as evidenced by Ramsden (Ramsden DA. Polymerases in nonhomologous end joining: building a bridge over broken chromosomes. Antioxid Redox Signal. 2011 Jun 15;14(12):2509-19. doi: 10.1089/ars.2010.3429. Epub 2010 Oct 28. PMID: 20649463; PMCID: PMC3113452).
Regarding claim 1, Larson teaches a method of simultaneously preparing a mixture of RNA and DNA targets in a single reaction volume for sequencing, wherein the method comprises the following steps:
(a) providing a sample in the single reaction volume (Fig 6; example 3 in para [0121-0124] for examples),
wherein the sample comprises both RNA and DNA targets (Fig 6; [0122] “isolated cell-free nucleic acid sample includes a mixture of cfDNA and cfRNA”);
(b) contacting the sample with a target-specific primer in the single vessel (Fig 6; [0123] biotin-labeled random hexamer primers comprising UMIs), under conditions that do not allow DNA denaturation (Fig. 6 ; Fig. 7A, DNAs are not denatured),
wherein the target-specific primer hybridizes to at least one RNA target (Fig 6; [0123]),
wherein the target-specific primer comprises a nucleic acid barcode ([0123] biotin-labeled random hexamer primers may also include a unique molecular identifiers (UMIs)),
wherein the nucleic acid barcode distinguishes cDNA molecules from DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA ([0123] only cDNA molecules are labeled with the UMI in 1st strand synthesis);
(c) extending the target-specific primer hybridized to the at least one RNA target with a nucleic acid polymerase in the single reaction volume,
wherein the nucleic acid polymerase has reverse transcriptase activity, thereby forming a cDNA strand (Fig 6; [0123] first strand cDNA synthesis from RNA template is via reverse transcriptase, see in [0023] lines 13-17, [0048] lines 1-12),
wherein the nucleic acid barcode is added to the cDNA strand by the reverse transcriptase activity ([0123]; [0023] lines 13-17, [0048] lines 1-12); and
(d) contacting the sample with: (i) an RNaseH, and (ii) at least one nucleic acid end repair enzyme, in the single reaction volume ([0124] DNA polymerase possesses end-repair function, see Ramsden in Abstract), thereby forming a mixture of double-stranded DNA and double-stranded cDNA ([0124] at the end of step 620, the reaction comprises a mixture of double-stranded DNA and double-stranded cDNA, as a result of 2nd strand synthesis; [0097], lines 7-8, cfDNA are double-stranded; see also [0101]).
Regarding the newly amended steps (a-d), they describe generating double-stranded cDNA from RNA in a sample comprising both RNA and DNA, in a single reaction volume. In other words, the claimed method performs double-stranded cDNA synthesis of RNA molecules while DNA is present in the background.
A skilled artisan would recognize that Example 3, as illustrated in Fig. 6 of Larson, anticipates this feature. Fig. 6 of Larson teaches, performing first strand and second strand cDNA synthesis of cfRNA in a cell-free nucleic acid sample includes a mixture of cfDNA and cfRNA.
It is evident that the cfDNA remains in the same single reaction mixture until after second strand synthesis. Specifically, in step 625 (following step 620, 2nd strand synthesis), biotinylated cDNA is captured using streptavidin beads, and the mixture is separated into a biotinylated cDNA fraction (derived from cfRNA) and a cfDNA supernatant fraction. Thus, the cfDNA is present in the single reaction mixture throughout all steps leading up to and including second-strand synthesis, as it must be present to allow the subsequent separation in step 625.
Regarding claim 5, Larson teaches a preliminary step of fragmenting the RNA and DNA targets ([0046] lines 11-16; [0109] physical fragmentation is applied to the sample comprising both RNA and DNA; [0060] lines 5-7, “In the first step, DNA is sheared into fragments of, or otherwise provided (e.g. as naturally occurring cfDNA molecules,”).
Regarding claim 6, Larson teaches contacting the mixture of double-stranded DNA and double-stranded cDNA with an adaptor, to form adapted DNA (Fig 2B; Fig. 4B for examples).
Regarding claim 8, Larson teaches barcode is a unique molecular identifier (UID) ([0097]lines 9-12, unique molecular identifier).
Regarding claim 9, Larson teaches amplifying (e.g.,[0054]; [0106]; [0113] PCR amplification).
Regarding claims 16-17, Larson teaches a single tube ([0082] line 11) 1.
Claims 1, 6, 8-9 and 16-17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Rooijers (Rooijers et al., Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells. Nat Biotechnol. 2019 Jul;37(7):766-772. doi: 10.1038/s41587-019-0150-y. Epub 2019 Jun 17. PMID: 31209373; PMCID: PMC6609448.) as evidenced by Invitrogen (SuperScript® Double-Stranded cDNA Synthesis Kit; Invitrogen by Life Technologies Corporation, 2011).
Regarding claim 1, Rooijers teaches a method of simultaneously preparing a mixture of RNA and DNA targets in a single reaction volume for sequencing (Fig. 1a), wherein the method comprises the following steps:
(a) providing a sample in the single reaction volume (Fig. 1a),
wherein the sample comprises both RNA and DNA targets (Fig. 1a);
(b) contacting the sample with a target-specific primer in the single vessel (Fig. 1a, mRNA primer), under conditions that do not allow DNA denaturation (Fig. 1a, DNA is not denatured),
wherein the target-specific primer hybridizes to at least one RNA target (Fig. 1a),
wherein the target-specific primer comprises a nucleic acid barcode (Fig. 1a, mRNA primer, sample barcode),
wherein the nucleic acid barcode distinguishes cDNA molecules from DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA (Fig. 1a; page 10, para 3, lines 5-6, “Raw reads were processed by demultiplexing on barcodes (simultaneously using
the DamID and transcriptomic barcodes), allowing no mismatches”);
(c) extending the target-specific primer hybridized to the at least one RNA target with a nucleic acid polymerase in the single reaction volume (Fig. 1a),
wherein the nucleic acid polymerase has reverse transcriptase activity, thereby forming a cDNA strand (Fig. 1a; page 9, lines 14-18),
wherein the nucleic acid barcode is added to the cDNA strand by the reverse transcriptase activity (Fig. 1a); and
(d) contacting the sample with: (i) an RNaseH (Page 9, line 20), and (ii) at least one nucleic acid end repair enzyme (Page 9, lines 14-20, Invitrogen’s cDNA synthesis system is used for 1st and 2nd strand DNA synthesis, which uses DNA Polymerase I and T4 DNA polymerase having end-repair function in 2nd strand synthesis step, see Invitrogen, page 3, "Second-Strand Synthesis"), in the single reaction volume, thereby forming a mixture of double-stranded DNA and double-stranded cDNA (Fig. 1a).
Regarding claim 6, Rooijers teaches contacting the mixture of double-stranded DNA and double-stranded cDNA with an adaptor, to form adapted DNA (Fig. 1a).
Regarding claim 8, Rooijers teaches a sample identifier (SID) (Fig. 1a).
Regarding claim 9, Rooijers teaches a step of amplifying a(Fig. 1a, linear amplification via IVT).
Regarding claims 16-17, Rooijers teaches a single tube (Fig. 1a, each well in a plate is a single tube).
Prior Art
For the purpose of compact prosecution, the examiner has reviewed the application's entire disclosure, but has not readily identified any subject matter that is not taught or suggested by the prior art, or combined in a non-obvious way.
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
The prior art has disclosed a large number of methods for producing a labeled cDNA product starting from RNA in the presence of double stranded DNA, in a single reaction mixture:
See Fig. 1 in WO2018126278A2 - Methods to distinguish rna and dna in a combined preparation; published 2018-07-05; cited as Foreign Pat Document #5 in IDS filed 06/07/2023;
See Fig. 1 in WO2018119452A2 - Methods and systems for analyzing nucleic acid molecules; published 2018-06-28; cited as Foreign Pat Document #4 in IDS filed 06/07/2023;
See Figure 1 in Dey et al., Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol. 2015 Mar;33(3):285-289. doi: 10.1038/nbt.3129. Epub 2015 Jan 19. PMID: 25599178; PMCID: PMC4374170; cited as Non-Pat lit Document #1 in IDS filed 06/07/2023;
See Figure 1 in Reuter et al. , Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling. Nat Methods. 2016 Nov;13(11):953-958. doi: 10.1038/nmeth.4028. Epub 2016 Oct 10. PMID: 27723755; PMCID: PMC5734913;
See Fig. 1 in Kong et al., Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures, Clinical Chemistry, Volume 65, Issue 2, 1 February 2019, Pages 272–281;
See Fig. 1b and "Cell sorting and lysis" section in Li et al., RNase H-dependent PCR-enabled T-cell receptor sequencing for highly specific and efficient targeted sequencing of T-cell receptor mRNA for single-cell and repertoire analysis. Nat Protoc. 2019 Aug;14(8):2571-2594. doi: 10.1038/s41596-019-0195-x. Epub 2019 Jul 24. PMID: 31341290; PMCID: PMC7189368.
Conclusion
No claims are 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 TIAN NMN YU whose telephone number is (703)756-4694. The examiner can normally be reached Monday - Friday 8:30 am - 5:30 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, Gary Benzion can be reached at (571) 272-0782. 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.
/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Moreover, using a tube to contain a reaction mixture cannot be the inventive concept as tubes are routinely used in performing molecular biology experiments to contain reactions, samples, and reagents. A skilled artisan would understand that, a reaction, unless specified otherwise, is typically performed in a reaction tube.
See Mohatt and Cripps ; Molecular Tools in the Classroom ; achieved on Wayback Machine May 04, 2017; note that all the basic molecular laboratory classroom experiments use tubes;
See also Korfhage et al., "Whole‐transcriptome amplification of single cells for next‐generation sequencing." Current Protocols in Molecular Biology 111.1 (2015): 7-20.; note that all library prep steps require performing reactions in tubes.
Prosecution Insights