DETAILED ACTION
Status of the Claims
Claims 1-19 are pending and examined herein.
Claims 1-3, 6, 8, 11-14, and 19 are amended.
The following Office Action is in response to Applicant's communication dated 06/09/2026. Rejection(s) and/or objection(s) not reiterated from previous office actions are hereby withdrawn. Specifically, the objections to the disclosure and claims 2, 3, 6, 8, 11, 13, and 14 for minor informalities as well as the rejections of claims 2, 3, 13, 14, and 19 under 35 U.S.C. 112(b) are withdrawn in light of Applicant's amendments. The following rejections(s) and/or objection(s) are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Interpretation
Claims 1 and 12 recite the limitation of a “single primer”. For the purposes of examination, this limitation is taken to mean one primer in sequence, though there can be multiple copies. Additionally, this limitation is interpreted as excluding the presence of additional primers configured to amplify the cDNA and transposed chromatin.
The preamble of claims 12-19 recite a “kit.” The specification, however, does not define this term, and so it is being interpreted to encompass any collection of reagents that includes all of the elements of the claims. Any further interpretation of the word is considered an “intended use” and does not impart any further structural limitation of on the claimed subject matter.
New Claim Rejections - 35 USC § 103
Necessitated by Amendment
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 12-19 are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. (Nat Struct Mol Biol, 2019, 26:1063-1070; of record), as evidenced by Oscorbin et al. (Computational and Structural Biology Journal, 2021, 19: 6315-6327; of record), in view of Picelli et al. (Nature Protocols, 2014, 9:171-181; of record).
Regarding claim 12, Zhu teaches a stabilizing agent (“RNase Inhibitor Mix” – Methods, col. 1, line 63), a tagmentation agent (“Tn5 transposomes” – Methods pg. 1, col. 1, line 33), an adapter (“DNA adaptor oligos” – Methods pg. 1, col. 1, line 34), wherein the Tn5 transposome complex/reagent is loaded with the adapter (“loaded transposases” – Methods pg. 1, col. 1, line 41), and a reverse transcriptase enzyme (“Maxima Reverse Transcriptase” – Methods pg. 1, col. 1, line 67). Additionally, Zhu discloses primers configured to amplify the of cDNA and the transposed chromatin (Supplemental Table 1: PA-R).
Zhu does not explicitly disclose that the reverse transcriptase enzyme is configured to transcribe DNA from both a DNA and an RNA template and perform a terminal transferase activity by template switching and an introduction of an adapter sequence into a cDNA and a transposed chromatin of the target. However, the properties recited (DNA- and RNA-dependent DNA-synthesis, terminal transferase activity via template switching) are inherent properties of Maxima Reverse Transcriptase (an MMLV reverse transcriptase). As stated in the instant specification, “RAM-seq was performed using the inherent terminal transferase and template switching capability of a Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.” Additionally, DNA- and RNA- dependent DNA-synthesis is a recognized inherent ability of MMLV reverse transcriptases, as evidenced in Oscorbin which states, “the reverse transcriptase of Moloney MuLV is a typical RT… possessing 3 catalytic activities: RNA- and DNA- dependent DNA polymerase, and RNAse H” (pg. 6316, “Properties”, lines 1-4). Because the claimed properties are inherent to the Maxima Reverse Transcriptase, Zhu also anticipates these properties (see MPEP § 2112).
Zhu also does not disclose a single primer configured to amplify the cDNA and the transposed chromatin in a single reaction, but does disclose one set of primers for the amplification of both cDNA and transposed chromatin (Figure 1a; Supplemental Table 1: PA-R). The difference between Zhu and the instant claim is therefore the use of different forward and reverse primers by Zhu. However, Picelli teaches a single primer configured to amplify cDNA in a single reaction (p. 172, col. 2, 2nd ¶: “PCR using a single primer”; Figure 1: “PCR preamplification”). Picelli, therefore, teaches the use of a single primer as both a forward and reverse primer. This indicates that the difference between the claimed invention and the prior art is the lack of actual combination of the elements taught by Zhu and Picelli. Additionally, adapter and primer design are routine tasks and therefore a person of ordinary skill in the art could have used known methods to combine the elements from Zhu and Picelli. Finally, each element merely performs the same function as it does separately, specifically PCR amplification. Based on this, it is clear that a person of ordinary skill in the art would have recognized that the results of the combination of two primers configured to amplify cDNA and transposed chromatin in a single reaction from Zhu with a single primer configured to amplify cDNA from Picelli would predictably result in a single primer configured to amplify both cDNA and transposed chromatin in a single reaction.
Regarding claim 13, Zhu further teaches that the adapter comprises a nucleic acid sequence at least 80% identical to the sequence set forth in SEQ ID NOs: 1-3 (Supplementary Table 1: SEQ ID NO 1 – AdapterA; SEQ ID NO 3 – pMENTs).
Regarding claim 14, Zhu teaches that the adapter comprises a nucleic acid sequence at least 80% identical to the sequence set forth in SEQ ID NOs: 4-6 (Supplementary Table 1: SEQ ID NO 3 – pMENTs).
Regarding claim 15, Zhu further discloses that the primer is configured to be introduced at the 3’ end of a first strand of the cDNA and a 3’ end of a chromatin of the target (Zhu Figure 1a-2, annotated below). However, Zhu does not disclose the primer being configured to be introduced at the 5’ end of a first strand of the cDNA.
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Zhu Figure 1a-2, annotated
Picelli teaches a primer configured to be introduced at both 5’ and 3’ ends of a first strand of DNA (Picelli Figure 1, annotated below). Picelli specifies that “every full-length cDNA carries … an additional artificial sequence, which in this case is the same as the one located at the 5′ end of the oligo-dT primer (i.e., the 3′-end of the mRNA)” (page 2, col. 2, lines 11-15).
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Picelli Figure 1, annotated
Based on the reasoning provided regarding claim 12, a person of ordinary skill in the art would have recognized that the results of the combination of a primer configured to be introduced at the 3’ ends of a cDNA and a chromatin from Zhu with a primer configured to be introduced at both 5’ and 3’ ends of a cDNA from Picelli would predictably result in a single primer configured to be introduced at all three locations for amplification of cDNA.
Regarding claim 16, Zhu further teaches the reverse transcriptase enzyme comprises a Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (“Maxima Reverse Transcriptase” – Methods pg. 1, col. 1, line 67).
Regarding claim 17, Zhu further teaches that the target comprises a nucleus, a whole cell, or a combination thereof (“nuclei” – Methods pg. 1, col. 1, line 52).
Regarding claim 18, Zhu teaches that the stabilizing agent is configured to stabilize the target by irreversibly inactivating RNases (see claim 12).
Regarding claim 19, Zhu teaches that the library simultaneously includes profiles of transcriptome and chromatin accessibility with the target (pg. 1063, col. 2, lines 8-10).
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Response to Arguments
Applicant’s arguments from 06/09/2026 argue that not all elements are taught. These arguments have been considered by are not persuasive.
Applicant argues that Zhu discloses tailing and adapter introduction by alternate means from the claimed reverse transcriptase and template switching. In response, it is noted that the features upon which applicant relies (performing tailing and adapter introduction via template switching using a reverse transcriptase) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Particularly, this claim is directed toward a composition (“kit”) and the recited properties of the reverse transcriptase (“configured to…”) are therefore considered an intended use.
A recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim. Furthermore, as per MPEP § 2115, “[i]nclusion of material or article worked upon by a structure being claimed does not impart patentability to the claims.” In re Young, 75 F.2d 996, 25 USPQ 69 (CCPA 1935) (as restated in In re Otto, 312 F.2d 937, 136 USPQ 458, 459 (CCPA 1963)), and that the manner or method in which a machine is to be utilized is not germane to the issue of patentability of the machine itself. See also MPEP § 2111.02, citing In re Sinex, 309 F.2d 488, 492, 135 USPQ 302, 305 (CCPA 1962) (statement of intended use in an apparatus claim did not distinguish over the prior art apparatus), and MPEP § 2112.01 citing In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990) stating that “discovery of an unobvious property and use does not overcome the statutory restraint of section 102 when the claimed composition is known”.
As the claimed properties of the reverse transcriptase are inherent to the disclosed MMLV transcriptase of Zhu, this indicates that the reverse transcriptase of Zhu is capable of performing the intended use and therefore meets the claim.
It is believed that Applicant’s remaining arguments have been sufficiently addressed by the modified rejections necessitated by amendment.
Modified Claim Rejections - 35 USC § 103
Necessitated by Amendment
Claims 1-9 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Zhu et al. in view of Ohtsubo et al. (Sci Rep, 2017, 7: 41769; of record) and Picelli et al.
Regarding claim 1, Zhu teaches a method comprising stabilizing a target (“RNase Inhibitor Mix” – Methods pg. 1, col. 1, line 63), performing a tagmentation on the target using a Tn5 transposase reagent, wherein the Tn5 transposase reagent is loaded with an adapter (pg. 1063, col. 2, lines 36-38), and generating a library using a reverse transcriptase enzyme (“Maxima Reverse Transcriptase” – Methods pg. 1, col. 1, line 67). Zhu additionally teaches performing a terminal transferase activity (Zhu Figure 1a-1, annotated below; pg. 1064, col. 1, lines 11-13), an introduction of an adapter sequence (“anchor oligo” – Methods pg. 1, col. 2, line 31) into a cDNA and a transposed chromatin of the target (Zhu Figure 1a), and amplifying the cDNA and transposed chromatin in a single reaction (Fig. 1a: “preamplification”). However, Zhu does not teach template switching by the reverse transcriptase enzyme to introduce the adapter sequence, nor does Zhu teach a single primer for the amplification.
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Zhu Figure 1a-1, annotated
Ohtsubo teaches that a reverse transcriptase can be configured to perform a template switching on both a cDNA (pg. 1, Introduction, paragraph 3) and blunt-end DNA, such as a transposed chromatin (pg. 7, Discussion, lines 1-2). Additionally, Ohtsubo teaches that the use of a reverse transcriptase for terminal transferase activity by template switching is preferable to that of other enzymes with terminal transferase activity due to its strong tailing activity, small tail length, and ability to append any nucleotide (pg. 7 and 9). Specifically, Ohtsubo writes, “the use of strong tailing activity reduces the amount of DNA needed for analysis, which will be useful for single-cell or ancient DNA analyzes” (pg. 9, lines 23-24). Furthermore, one of ordinary skill in the art would have had a reasonable expectation of success in using a reverse transcriptase to perform a terminal transferase activity on both a cDNA and a transposed chromatin because reverse transcriptases are routinely used in similar contexts (pg. 1, Introduction, paragraph 3).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the reverse transcriptase configured to perform a terminal transferase activity by a template switching from Ohtsubo to the method of Zhu with a reasonable expectation of success, based on the motivation taught in Ohtsubo.
Zhu also does not disclose a single primer for amplifying the cDNA and the transposed chromatin in a single reaction. However, this limitation is rendered obvious by the disclosure of Picelli, as discussed regarding claim 12.
Regarding claim 2, Zhu further teaches that the adapter comprises a nucleic acid sequence at least 80% identical to the sequence set forth in SEQ ID NOs: 1-3 (Supplementary Table 1: SEQ ID NO 1 – AdapterA; SEQ ID NO 3 – pMENTs).
Regarding claim 3, Zhu also teaches that the adapter comprises a nucleic acid sequence at least 80% identical to the sequence set forth in SEQ ID NOs: 4-6 (Supplementary Table 1: SEQ ID NO 3 – pMENTs).
Regarding claim 4, Zhu additionally discloses placing the target on a solution, wherein the solution comprises an effective amount of phosphate-buffered saline (Methods pg. 1, col. 1, line 63).
Regarding claim 5, Zhu additionally discloses freezing the target before generating the library (p. 1069, col. 1, line 20).
Regarding claim 6, Zhu further discloses performing reverse transcription on the target (pg. 1063, col. 2, lines 15-16) and performing PCR amplification on the cDNA and the chromatin of the target at a predetermined cycle (pg. 1063, col. 2, lines 16-19). A primer for amplification introduced at both 5’ and 3’ ends of a first strand of the cDNA and a 3’ end of the chromatin of the target is discussed in view of Zhu and Picelli in the previous section regarding claim 15. Furthermore, Zhu discloses splitting the amplified cDNA and chromatin into a first group for an RNA library and a second group for an ATAC library (pg. 1064, col. 1, lines 13-17).
Regarding claim 7, Zhu further discloses generating the library comprises generating the RNA library by performing an RNA sequencing on the first group (Methods pg. 1, col. 2, lines 61-63), wherein the first group for the RNA library comprises a cell-specific barcode introduced through an RT primer (“barcoded RT primers” – Methods pg. 1, col. 1, line 65) and generating the ATAC library by performing an ATAC sequencing on the second group (Methods pg. 1, col. 2, lines 61-63), wherein the second group for the ATAC library comprises a cell-specific barcode introduced through an indexing primer (“Indexing PCR” – Methods, col. 2, lines 55-61).
Regarding claim 8, Zhu further teaches the reverse transcriptase enzyme comprises a Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (“Maxima Reverse Transcriptase” – Methods pg. 1, col. 1, line 67).
Regarding claim 9, Zhu further teaches that the target comprises a nucleus, a whole cell, or a combination thereof (“nuclei” – Methods pg. 1, col. 1, line 52).
Regarding claim 11, Zhu teaches that the library simultaneously includes profiles of transcriptome and chromatin accessibility with the target (Introduction, col. 2, lines 8-10).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Zhu in view of Ohtsubo et al. and Picelli et al., and further in view of Nichols et al. (Current Protocols in Molecular Biology, 2008, 84:3.13.1-3.13.8; of record).
See the previous section for prior art analysis of the limitations of claim 1. Additionally, Zhu discloses lysing the target (pg. 1064, col. 1, lines 13-17) and stabilizing the target using a solution that irreversibly inactivates RNases (“RNase Inhibitor Mix” – Methods pg. 1, col. 1, line 52). The distinction between Zhu and the instant claim is Zhu used a restriction enzyme digestion step while the claim specifies the use of RNases. However, the result of both steps is still the lysing of the target, indicating that the only difference is in the agent used.
Furthermore, Nichols teaches that RNases are commonly used to hydrolyze RNA to remove it from DNA preparations. Therefore, one of ordinary skill in the art before the time of the effective filing date of the claimed invention could have substituted the restriction enzyme digestion step of Zhu with a lysing using RNases, yielding the predictable result of lysing the target.
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Response to Arguments
Applicant's arguments filed 06/09/2026 have been fully considered but they are not persuasive. Applicant argues that there is insufficient motivation to combine the disclosures of Ohtsubo and Zhu and that Ohtsubo teaches away from the claimed features.
Applicant argues that the motivation from Ohtsubo that reverse transcriptases have “strong tailing activity” that “reduces the amount of DNA needed for analysis” does not provide motivation for the claimed function of performing template switching and introduction of an adapter sequence. In response, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). The function of template switching in high-throughput sequencing applications is to append additional sequences, such as adapter sequences, to a target. Indeed, Zhu discloses that “DNA barcode tags are first added to genomic DNA and cDNA by TDT-assisted DNA tailing” (Fig. 1a description). Therefore, one of ordinary skill in the art would understand that using MMLV-RT to perform DNA tailing would also lead to using MMLV-RT for template switching and introduction of an adapter sequence, as these events are all precipitated by the tailing action.
Applicant further states that “Ohtsubo teaches away from the claimed features”, but does not provide evidence of this. This is a conclusory statement and is not considered persuasive.
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.
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/ALEXANDRA OLSON/Examiner, Art Unit 1684
/JEREMY C FLINDERS/Primary Examiner, Art Unit 1684
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