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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed on July 07, 2026 in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on June 17, 2026 has been entered.
Status of Claims / Response to Amendment
This office action is in response to an amendment filed on June 17, 2026.
Claims 1, 5-6, 8-9 and 16-17 were previously pending. Applicant amended claims 1, 5, and 8.
Claims 1, 5-6, 8-9 and 16-17 are currently pending and under consideration.
All of the previously presented rejections have been withdrawn as either being addressed or obviated by the amendment of the claims, which introduces a new combination of elements that were not previously considered in the prior rejection.
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.
Claim Objections
Claim 1 is objected to because of the following informalities:
In claim 1, lines 5-6, to improve consistency within the claim, "wherein the cellular DNA targets and the RNA targets are from a sample" should read:
"wherein the cellular DNA targets and the cellular RNA targets are from a sample," to properly refer back to "cellular RNA targets" first introduced in claim 1, line 5.
In claim 1, part (a) lines 1-2, "wherein the sample comprises both the target cellular RNA targets and cellular DNA targets" should read:
"wherein the sample comprises both the
In claim 1, part (d) lines 3-4, to improve consistency within the claim, "wherein the double-stranded cDNA comprises the nucleic acid barcode, and the double-stranded DNA lacks the barcode" should read:
"wherein the double-stranded cDNA comprises the nucleic acid barcode, and the double-stranded DNA lacks the nucleic acid barcode." 1
Priority
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.
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 purpose of applying prior art, claim 1 has been amended to recite "cellular RNA targets" and "cellular DNA targets." These terms are not expressly defined in the specification.
The disclosure does not define any feature that distinguishes a DNA target or RNA target from any other DNA or RNA molecule.
Therefore, under BRI, "cellular RNA targets" and "cellular DNA targets" are interpreted to encompass any DNA or RNA originating from a cellular biological source. This includes cell-free or circulating nucleic acids released from cells 2.
Claim 1 has been amended to recite "a DNA library suitable for sequencing." The claim then defines the DNA library as "comprises a mixture of double-stranded DNA and double- stranded cDNA."
The claim does not recite any particular sequencing technology, and the specification does not provide any definition of "DNA library suitable for sequencing" or identify any additional structural, compositional features required for such suitability.
Accordingly, under BRI, a DNA library "suitable for sequencing" in the claim is interpreted to encompass any DNA library comprising a mixture of double-stranded DNA and double- stranded cDNA.
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. cellular RNA target). Therefore, random primers are encompassed by this term, as they hybridize to various regions on target nucleic acids 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])
Regarding claim 9 reciting "optionally sequencing the adapted DNA." The term "optional" is interpreted according to its ordinary meaning as identifying a feature that is permitted but not required.
Response to Arguments
Applicant's arguments filed on June 17, 2026 have been fully considered.
Claim Rejections - 35 USC § 102
In the prior Office Action (Final Office Action- 04/21/2026):
Claims 1, 5-6, 8-9 and 16-17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Larson, as evidenced by Ramsden;
Claims 1, 6, 8-9 and 16-17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Rooijers as evidenced by Invitrogen.
The prior rejections set forth above have been withdrawn as being obviated by the amendment of the claims, which added new limitations to the claims, that were not considered in the previous rejections.
However, as discussed in detail in the new grounds of rejections below, the claims are still considered obvious in view of Larson and additional prior art references; and anticipated by Rooijers, with additional evidenced and support provided in the rejection to address the newly amended claims.
To the extent applicable to the current rejections set forth in this Office Action, Applicant's arguments are addressed below.
Regarding Larson, Applicant asserts that it does not teach or suggest the claim as amended:
"Larson does not disclose, teach, or suggest, inter alia, a method for generating a DNA library suitable for sequencing, comprising a mixture of double-stranded DNA and double-stranded cDNA, which originate from cellular DNA targets and cellular RNA targets, respectively, from a sample, in a single reaction volume, as is recited in the claimed methods, as amended. (Remarks, page 9-10).
Specifically, Applicant asserts that Larson teaches preparing DNA and RNA targets in separate reaction volumes, citing Example 3, which states "two separate sequencing libraries are prepared," (illustrated in Fig. 6) for support (Remarks, page 11-13).
Applicant's observation regarding Example 3 is acknowledged. Example 3 in Larson discloses,
a method of preparing cell-free nucleic acid libraries using biotin-labeled random hexamer primers to tag cDNA reverse transcribed from cfRNA in a cell-free nucleic acid Sample ([0121]). The reverse transcription and second strand synthesis steps are carried out in a cell-free nucleic acid sample that includes a mixture of cfDNA and cfRNA ([0122]-[0123]; [0130]).
After second strand synthesis, the biotin label incorporated into double stranded cDNA derived from cfRNA is used to separate the cell-free nucleic acid sample into a cfDNA fraction and a cDNA fraction (derived from cfRNA) for preparation of two separate sequencing libraries. ([0131]-[0133])
However, Applicant's broader assertion that Larson does not teach or suggest generating a single sequencing library comprising both DNA and cDNA in a single reaction volume is not persuasive because it overgeneralizes the teachings of Larson and overlooks Larson's disclosure in other embodiments.
For clarity of record, Larson expressly teaches preparing a single sequencing library comprising both DNA and cDNA in Examples 1 and 2.
Larson teaches in Example 1:
"[0106] At step 135, a sequencing library 270 is prepared. For example, a sequencing library preparation protocol (e.g., TruSeq® library preparation protocol (Illumina, Inc.)) that includes the steps of end repair (not illustrated), 3′ end A-tailing (not illustrated), ligation of sequencing adapters 275, and PCR amplification (not illustrated) is used to prepare sequencing library 270. Sequencing library 270 includes cfDNA amplicons 280 and cDNA amplicons 285 (derived from cfRNA 220). cDNA amplicons 285 include barcode region 245 and universal primer region 250. "
Larson teaches in Example 2:
"[0119] In step 335, a sequencing library 465 is prepared. For example, a sequencing library preparation protocol (e.g., TruSeq® library preparation protocol (Illumina, Inc.)) that includes the steps of end repair (not illustrated), 3′ end A-tailing (not illustrated), ligation of sequencing adapters 470, and PCR amplification is used to prepare sequencing library 465. Because of nick 460 in cfRNA/cDNA hybrid molecules 455, cfRNA strand 420 is not amplified during the PCR amplification step of the illustrated method. Sequencing library 465 includes cfDNA amplicons 475 and cDNA amplicons 480 (derived from cfRNA 420). cDNA amplicons 480 include barcode region 430 and universal primer region 435."
Regarding Rooijers, Applicant asserts that it does not anticipate claim 1 because:
"Rooijers appears to teach that all target molecules have barcodes attached-one of two different types of barcodes-one type, DamID adapters binding to genomic DNA, and one type, CEL-Seq RT primers binding to mRNA. See, Rooijers, supra, at 10, 3rd para; see, also, id. at FIG. la." (Remarks, page 15-16).
This argument relates to the newly added limitation "wherein the double-stranded cDNA comprises the nucleic acid barcode, and the double-stranded DNA lacks the barcode."
Here, "the barcode" is interpreted as referring to, and having the same meaning as, "the nucleic acid barcode." As noted in the claim objection, the claim does not previously recite any other barcode besides the "nucleic acid barcode." Thus, "the barcode" should read "the nucleic acid barcode" to properly refer back to the "nucleic acid barcode," first introduced in part (b) of claim 1, reciting: "wherein the one or more target-specific primers hybridizes to one or more cellular RNA targets, wherein each target-specific primer comprises a nucleic acid barcode."
Applicant's argument has been considered but is not persuasive. The claim defines the "nucleic acid barcode" as being comprised by "target-specific primer," and added to cDNA by reverse transcription (i.e., "wherein the nucleic acid barcode is added to the cDNA strand by the reverse transcriptase activity"), and "wherein the nucleic acid barcode distinguishes the double-stranded cDNA molecules from double-stranded DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA." Accordingly, the RNA-specific barcode comprised by the RT primer in Rooijers meets the claimed description for “nucleic acid barcode,” whereas the DNA-specific barcode, comprised by an adapter in Rooijers does not. Therefore, the DNA molecule in Rooijers's library does not comprise the claimed "nucleic acid barcode."
As currently written and under BRI, the claim does not exclude the DNA molecule from having ANY label or barcode. Rather, the claim requires only that the DNA molecule lacks the nucleic acid barcode added to cDNA by reverse transcription. Rooijers teaches this feature.
It is further noted that the application's disclosure does not appear to support a sequencing library in which the DNA molecule has no barcode at all. Instead, the specification teaches contacting DNA with barcoded adapters, which is consistent with the teaching of Rooijers:
"In some embodiments, the method further comprising contacting the mixture of double-stranded DNA and double-stranded cDNA with an adaptor having one or more barcodes, e.g., a unique molecular identifier (UID) and a sample identifier (SID)." ([0005] lines13-16)
"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." ([0036] lines 4-7)
"In some embodiments, the invention comprises a step of adaptor ligation. The adaptor may be ligated to the ends of a double stranded DNA molecule formed as described herein." ([0040]lines 1-3).
Claim Rejections - 35 USC § 102 -- New
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, 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).
Rooijers teaches a methods of simultaneous processing of genomic DNA and mRNA from a single cell for sequencing library generation, wherein the library comprises both RNA and DNA sequences (Figure 1a and methods).
Regarding claim 1, Rooijers teaches a method of generating a DNA library suitable for sequencing (Figure 1a),
wherein the DNA library comprises a mixture of double-stranded DNA and double-stranded cDNA (Figure 1a),
wherein:
(i) the double-stranded DNA originates from cellular DNA targets (Figure 1a), and
(ii) the double-stranded cDNA originates from cellular RNA targets (Figure 1a),
wherein the cellular DNA targets and the RNA targets are from a sample (Figure 1a),
wherein the method comprises the following steps, which are all conducted in a single reaction volume:
(a) providing the sample in the single reaction volume (Fig. 1a, a single reaction well),
wherein the sample comprises both the target cellular RNA targets and cellular DNA targets (Fig. 1a);
(b) contacting the sample with one or more target-specific primers in the single reaction volume (Fig. 1a, mRNA primer),
under conditions that do not allow DNA denaturation (Fig. 1a, DNA is not denatured),
wherein the one or more target-specific primers hybridizes to one or more cellular RNA targets (Fig. 1a),
wherein each target-specific primer comprises a nucleic acid barcode (Fig. 1a, mRNA primer, sample barcode),
wherein the nucleic acid barcode distinguishes the double-stranded cDNA molecules from double-stranded DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA (Fig. 1a, the cDNA and genomic DNA are barcoded using unique, predefined sample barcodes, in separate steps using different chemistries; 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”; see also page 10, para 1-2);
(c) extending the one or more target-specific primers hybridized to the one or more cellular RNA target with one or more nucleic acid polymerases in the single reaction volume (Fig. 1a),
wherein the nucleic acid polymerase has reverse transcriptase activity,
thereby forming one or more cDNA strands (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 the mixture of double- stranded DNA and double-stranded cDNA (Fig. 1a),
wherein the double-stranded cDNA comprises the nucleic acid barcode, and the double-stranded DNA lacks the barcode (Fig. 1a, the genomic DNA library (labeled by DamID adapters) does not have the same barcode as the RNA library (Labeled by CEL-Seq primers), which is how DNA and RNA sequences can be distinguished in data processing), and thereby generating the DNA library.
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).
Claim Rejections - 35 USC § 103 -- New
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 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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 5-6, 8-9 and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Larson (US20180002749A1 - Differential tagging of rna for preparation of a cell-free dna/rna sequencing library; Published on 2018-01-04), in view of
Alpern (Alpern et al. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol 20, 71 (2019).; doi.org/10.1186/s13059-019-1671-x);
Enderle (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); and
Kennedy (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);
as evidenced by Illumina TruSeq (TruSeq_DNA_SamplePrep_Guide_15026486_C; Published 2012).
A) Larson teaches methods of distinguishing sequences of RNA and DNA in a sample, and that “analyzing both RNA and DNA from a single sample provides additional information than analyzing either one alone.” ([0029] lines 1-5).
Larson teaches “In general, distinguishing RNA from DNA involves differential treatment of one or both with respect to the other.” ([0029] lines 20-24) And further provides the example that “differential treatment can comprise association of a tag sequence specifically with RNA of a sample, or specifically with molecules derived from RNA of a sample (e.g. cDNA).” ([0029] lines 29-34).
Regarding claim 1, Larson teaches a method of generating a DNA library suitable for sequencing (Fig. 1; Fig 3),
wherein the DNA library comprises a mixture of double-stranded DNA and double-stranded cDNA (Fig. 1; [0099] “In a step 135, a sequencing library is prepared… The sequencing library now includes
amplicons from cfDNA and barcoded cDNA ( derived from cfRNA)” ; see also Fig 3),
wherein:
(i) the double-stranded DNA originates from cellular DNA targets ([0099] “cfDNA” cfDNAs are derived from cells, see claim interpretation ), and
(ii) the double-stranded cDNA originates from cellular RNA targets ([0099] “barcoded cDNA (derived from cfRNA)” cfRNAs are derived from cells, see claim interpretation),
wherein the cellular DNA targets and the RNA targets are from a sample (Fig. 1; [0094] “blood sample is obtained and circulating cell-free nucleic acids are isolated from the plasma fraction. The isolated cell-free nucleic acid sample includes a mixture of cfDNA and cfRNA.”),
wherein the method comprises the following steps, which are all conducted in a single reaction volume:
(a) providing the sample in the single reaction volume ([0094]),
wherein the sample comprises both the target cellular RNA targets and cellular DNA targets ([0094]);
(b) contacting the sample with one or more target-specific primers in the single reaction volume ([0095]; [0102] “At step 115, a random hexamer primer 225 is used in a reverse transcription reaction to synthesize first strand cDNA”; FIG. 2A) , under conditions that do not allow DNA denaturation (FIG. 2A, step 115, DNA is not denatured),
wherein the one or more target-specific primers hybridizes to one or more cellular RNA targets (FIG. 2A, step 115),
wherein the nucleic acid barcode distinguishes the double-stranded cDNA molecules from double-stranded DNA molecules in the mixture of double-stranded DNA and double-stranded cDNA ([0105] “Cell-free nucleic acid sample 210 now includes a mixture of cfDNA 215 and a population of double stranded cDNA molecules 265 ( derived from cfRNA 220) that are tagged with barcode region 245.”);
(c) extending the one or more target-specific primers hybridized to the one or more cellular RNA target with one or more nucleic acid polymerases in the single reaction volume (FIG. 2A, step 115; [0095]; [0102] extending RT primer using reverse transcriptase to transcribe cDNA from cfRNA in cell-free nucleic acid sample. Cell-free nucleic acid sample 210 now includes a mixture of cfDNA 215 and short cfRNA/cDNA hybrid molecules 230),
wherein the nucleic acid polymerase has reverse transcriptase activity (FIG. 2A, step 115; [0095]; [0102]) , thereby forming one or more cDNA strands (FIG. 2A, step 115; [0095]; [0102]); and
(d) contacting the sample with:
(i) an RNaseH (FIG. 2A, step 120; [0096] “In a step 120, the cfRNA in the cfRNA/cDNA hybrid molecules is degraded using, for example, excess RNase H. The cell-free nucleic acid sample now includes a mixture of cfDNA and first strand cDNA fragments that have a hydroxyl group on the 3' end of the molecule.”; [0103]), and
(ii) at least one nucleic acid end repair enzyme, in the single reaction volume ([0099] and [0106] preparing sequencing library including end repair using TruSeq® library preparation protocol (Illumina, Inc.), which is an enzymatic step (see Illumina TruSeq , p. 15, line 3), thus at least one enzyme is involved.), thereby forming the mixture of double- stranded DNA and double-stranded cDNA ([0099] The sequencing library now includes amplicons from cfDNA and barcoded cDNA ( derived from cfRNA).),
wherein the double-stranded cDNA comprises the nucleic acid barcode, and the double-stranded DNA lacks the barcode, and thereby generating the DNA library ([0099]; [0106] “Sequencing library 270 includes cfDNA amplicons 280 and cDNA amplicons 285 (derived from cfRNA 220). cDNA amplicons 285 include barcode region 245 and universal primer region 250”).
As detailed above, Larson teaches, in Example 1 (illustrated in FIG.1 and FIG. 2, described in [0093]-[0106] ), a method of preparing a sequencing library comprising DNA amplicons and barcoded, double stranded cDNA amplicons (see FIG. 2B, step 270), wherein the entire method is carried out in a single reaction comprising a mixture of RNA and DNA, or their products thereof. The fact that Larson teaches a library preparation method in which the RNA and DNA are mixed together in the same reaction (rather than separated), is supported by the explicitly descriptions in para. [0093] –[0106], wherein after each step, the sample is described as including a mixture of DNA and RNA, or their respective products. For examples:
“[0098] In a step 130, second strand cDNA is synthesized. For example, second strand cDNA is synthesized in an extension reaction using the universal primer sequence on the universal ligation adapter as a primer. The cell-free nucleic acid sample now includes cfDNA and double stranded cDNA (derived from cfRNA) that is tagged with a unique barcode.”
“[0106] At step 135, a sequencing library 270 is prepared. For example, a sequencing library preparation protocol ( e.g., TruSeq® library preparation protocol (Illumina, Inc.)) that includes the steps of end repair (not illustrated), 3' end A-tailing (not illustrated), ligation of sequencing adapters 275, and PCR amplification (not illustrated) is used to prepare sequencing library 270. Sequencing library 270 includes cfDNA amplicons 280 and cDNA amplicons 285 (derived from cfRNA 220). cDNA amplicons 285 include barcode region 245 and universal primer region 250.”
Larson in Example 1 teaches performing reverse transcription (RT) using RT primers (e.g., random hexamers), then differentially tag cDNA (reverse transcribed from RNA) with barcode via the mechanism of single-strand DNA ligation ([0093]).
Although this differs from the claimed approach, in which the RT primer itself comprises the barcode and adds the barcode to cDNA during reverse transcription, the difference is an obvious variation. Barcoding cDNA using barcoded RT primers was well-known in the art. A skilled artisan would have recognized post-RT barcode ligation (disclosed in Larson) and barcode incorporation during RT (in claim 1) are alternative, known approaches for differentially tagging cDNA.
cDNA tagging using barcoded RT primers is well-known in the art, this is supported by Alpern (see Figure 2), Enderle (See FIG.1) and Kennedy (See FIG. 1).
Specifically, Enderle (See FIG.1; [0054]) and Kennedy (See FIG. 1; [000119]; [000121]) both teach differentially tagging cDNA during reverse transcription using barcoded RT primers, in a mixture comprising both RNA and DNA, so that RNA-derived cDNA can be distinguished from DNA in sequencing.
Accordingly, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to modify Example 1 of Larson by using barcoded RT primers to tag cDNA during the reverse transcription step, instead of first generating cDNA and then ligating a barcode to the cDNA. This modification represents a simple substitution of one known cDNA-barcoding approach for another to yield the predictable result of cDNA that is differentially tagged from DNA. see MPEP 2141.
A skilled artisan would have been motivated to make this modification because adding the barcode during reverse transcription combines 1st strand cDNA synthesis and cDNA barcoding into a single step, thereby simplifies the workflow.
The person of ordinary skill would have had a reasonable expectation of success because barcoded RT primers were well known for cDNA tagging, as supported by Alpern (see Figure 2), Enderle (See FIG.1) and Kennedy (See FIG. 1). Enderle and Kennedy specifically demonstrate this approach in the same context as Larson, namely analyzing both DNA and RNA from a single sample while distinguishing RNA-derived cDNA from DNA using differential tags.
B) Regarding claim 5, Larson teaches a preliminary step prior to step (a), 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,”; [0044] lines 1-3, 24-29; “RNA is subjected to fragmentation, such as prior to joining tag oligonucleotides to the RNA, or prior to reverse transcription… In some embodiments, RNA species are the longest polynucleotide species in a sample, such that conditions that would be likely to fragment longer DNAs nonetheless preferentially fragment RNAs due to the higher likelihood of a fragmentation event occurring in the longer species.” Thus, a skilled artisan would readily appreciate that a preliminary fragmentation step prior to RT, applied to a sample comprising both RNA and DNA, would have fragmented both although at different degrees due to size difference).
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 ).
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).
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Reuter (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) in Figure 1 teaches generating, in a single reaction volume, a DNA library comprising double-stranded cDNA and DNA that are differentially tagged, and sequencing the DNA library and generating reads that comprise RNA-tag and DNA-tag.
Conclusion
Claim 1 is objected to; claims 1, 5-6, 8-9 and 16-17 are rejected. No claims are allowed.
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.
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/TIAN NMN YU/Examiner , Art Unit 1681
1 This amendment is suggested because the claim does not previously recite any other barcode besides the "nucleic acid barcode." Thus, "the barcode" should read "the nucleic acid barcode" to properly refer back to the "nucleic acid barcode," first introduced in part (b) of claim 1.
2 See page 426 in Schwarzenbach et al. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11, 426–437 (2011). doi.org/10.1038/nrc3066:
" The release of nucleic acids into the blood is thought to be related to the apoptosis and necrosis of cancer cells in the tumour microenvironment. Secretion has also been suggested as a potential source of cfDNA (Fig. 1). Necrotic and apoptotic cells are usually phagocytosed by macrophages or other scavenger cells8. Macrophages that engulf necrotic cells can release digested DNA into the tissue environment. In vitro cell culture experiments indicated that macrophages can be either activated or dying during the process of DNA release8. Fragments of cellular nucleic acids can also be actively released9,10. It has been estimated that for a patient with a tumour that weighs 100 g, which corresponds to 3 × 1010 tumour cells, up to 3.3% of tumour DNA may enter the blood every day11. On average, the size of this DNA varies between small fragments of 70 to 200 base pairs and large fragments of approximately 21 kilobases12. Tumour cells that circulate in the blood, and micrometastatic deposits that are present at distant sites, such as the bone marrow and liver, can also contribute to the release of cfNA13,14."
Prosecution Insights