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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on November 7th, 2022 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-15 and 32, in the reply filed on October 6th, 2025 is acknowledged.
Claims 33, 40, 48, and 73 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on October 6th, 2025.
Claim Summary
Claims 4-5, 7, 12, 14-15, 32, 48, and 73 have been amended. Claims 16-31, 34-39, 41-47, and 49-72 have been canceled. Claims 1-15, 32-33, 40, 48, and 73 are pending. Claims 33, 40, 48, and 73 are withdrawn from consideration as being drawn to a non-elected invention/species. Claims 1-15 and 32 are under examination and discussed in this Office action.
Claim 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.
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 8-11, 14, and 32 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 8 recites the limitation “wherein the reverse transcriptase further reverse transcribes the adjacent annealing site thereby replacing the 5' end of the adjacent fragment and creating excess single-stranded DNA”. However, this recitation does not include a step of halting the RT reaction. As currently written, it could be interpreted that the reaction is continuously occurring. If this is indeed the case, it is therefore unclear how the reaction is halted to allow for the later steps of trimming and ligating if the reverse transcriptase is still active and capable of displacing any newly synthesized cDNA it runs into. Claims 9-11 are also rejected here for their dependence on claim 8 and not further clarifying the identified issue. Turning to the specification, it appears that the reverse transcriptase eventually stops and falls off the template first before excess single-stranded cDNA is trimmed and ligated. For the purpose of compact prosecution, it is interpreted that the reverse transcriptase stops and falls off the template before trimming and ligating.
Claim 8 also recites the limitation “the adjacent fragment”. There is insufficient antecedent basis for this limitation in the claim. No claim from which claim 8 depends introduces an adjacent fragment. Claims 9-11 are also rejected here for their dependence on claim 8 and not further clarifying the identified issue.
Finally, Claim 8 recites the limitation “the reverse transcriptase further reverse transcribes the adjacent annealing site thereby replacing the 5’ end”. There is insufficient antecedent basis for this limitation in the claim. In claim 7, it is introduced that the reverse transcriptase reverse transcribes between two adjacent annealing sites. It is then unclear what constitutes the adjacent annealing site if two were previously introduced. Claims 9-11 are also rejected here for their dependence on claim 8 and not further clarifying the identified issue. For the purpose of compact prosecution, “the reverse transcriptase further reverse transcribes the adjacent annealing site thereby replacing the 5’ end” is interpreted to mean the reverse transcriptase further reverse transcribes an adjacent annealing site of the two adjacent annealing sites that is furthest downstream in the direction of reverse transcription.
Claim 14 recites the limitation “wherein the adapter primer further comprises… (b) a region complementary to a second unique molecular identifier (UMI-B)”. It is unclear from this recitation how there may be a region complementary to a second UMI when there is no second UMI claimed to be in the cDNA produced in claim 12. It is further unclear if the adapter primer is then intended to introduce a UMI-B, or if the cDNA should already have a UMI-B that the adapter primer will hybridize with. For the purpose of compact prosecution, the recitation “wherein the adapter primer further comprises… (b) a region complementary to a second unique molecular identifier (UMI-B)” will be interpreted as wherein the adapter primer further comprises… (b) a second unique molecular identifier (UMI-B).
Claim 32 recites the limitation "amplifying the double-stranded cDNA molecule via a polymerase chain reaction using a first primer and a second primer that are complementary to the first generic primer region and the second generic primer region, respectively" in step (b). There is insufficient antecedent basis for this limitation in the claim. Claim 1 and claim 12, from which claim 32 depends, do not introduce “a first generic primer region” or “a second generic primer region”. These terms are introduced in claim 4 and claim 14, respectively, in association with other aspects of the RT primer and adapter primer.
Claim Rejections - 35 USC § 102
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1 and 5-8 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Seitz (US 20170342409 A1).
Regarding instant claim 1, Seitz teaches a method of generating a DNA/RNA duplex from a target RNA molecule, comprising incubating a plurality of reverse transcriptase primers (RT primers) and the target RNA molecule under conditions such that the target RNA molecule is reverse transcribed generating a DNA/RNA duplex (Page 2, paragraph [0020]; Figure 1; Page 5, paragraph [0057]; Figure 2), wherein the plurality of RT primers are complementary to multiple annealing sites of the target RNA molecule such that each RT primer has an annealing site that is different than the annealing site of another RT primer in the plurality (Page 2, paragraph [0020]; Figure 1; Page 5, paragraph [0057]; Pages 5-6, paragraph [0063]; Figure 2).
Regarding instant claim 5, Seitz teaches the method of claim 1, wherein the target RNA molecule is reverse transcribed via a reverse transcriptase (Page 2, paragraph [0015]; Page 6, paragraph [0066].
Regarding instant claim 6, Seitz teaches the method of claim 5, wherein the reverse transcriptase is a processive reverse transcriptase like AMV RT (Page 12, paragraph 0122]) As evidenced by Promega (Choosing the Right Reverse Transcriptase [online]. Promega, [2025] [retrieved on December 4th, 2025]. Retrieved from: https://www.promega.com/resources/pubhub/choosing-the-right-reverse-transcriptase/), AMV RT is considered a processive reverse transcriptase (Page 2, paragraph 3).
Regarding instant claim 7, Seitz teaches the method of claim 5, wherein the reverse transcriptase reverse transcribes the sequence of the target RNA molecule between two adjacent annealing sites thereby generating complementary DNA fragments annealed to the target RNA molecule (Page 2, paragraph [0020]; Figure 1; Page 5, paragraph [0057]; Page 5, paragraph [0059]; Pages 5-6, paragraph [0063]; Figure 2).
Regarding instant claim 8, Seitz teaches the method of claim 7, wherein the reverse transcriptase further reverse transcribes the adjacent annealing site thereby replacing the 5' end of the adjacent fragment and creating excess single-stranded DNA (Page 2, paragraph [0020]; Figure 1).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 2 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Seitz (US 20170342409 A1).
Regarding instant claim 2, Seitz teaches the method of claim 1. Seitz does not directly teach wherein the sequence of the target RNA molecule between two adjacent annealing sites is 1,000 to 7,000 nucleotides long. However, Seitz does teach that depending on the concentration of the random primer, the average length of the product nucleic acids can be influenced (Page 12, paragraph [0130]). Furthermore, Seitz teaches that increasing the concentration of random primers can decrease the size of the elongated product (Page 12, paragraph [0130]), which also indicates that decreasing concentration increases elongated product size. Given that Seitz teaches RT stops when it reaches an adjacent downstream oligo stopper (Page 5, paragraph [0057]; Page 5, paragraph [0059]; Pages 5-6, paragraph [0063]; Figure 2), the length of a given elongated cDNA product inherently reflects the number of nucleotides between annealing sites in the target RNA. Therefore, with Seitz’s teaching that concentration of random primers reflects how long of a cDNA is produced, it would amount to routine optimization of the concentration of random primers to arrive at the claimed number of nucleotides between adjacent annealing sites. It is noted that the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 II. Thus, the claimed range merely represents routine optimization of the cited prior art.
Regarding instant claim 3, Seitz teaches the method of claim 2. Seitz further teaches wherein the sequence of the target RNA molecule between two adjacent annealing sites is about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, or 7,000 nucleotides long (see claim 2 for citations and 103 analysis, which are also applicable here).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Seitz (US 20170342409 A1), as applied to claims 1 and 5-8 above, in view of Vollmers (Genetic measurement of memory B-cell recall using antibody repertoire sequencing, PNAS, July 2013, 1120, 13463-13468).
Regarding instant claim 4, Seitz teaches the method of claim 1. Seitz further teaches that any one of the primers may comprise a sequence tag with a unique, preselected nucleic acid sequence for recognition of the sequence labelled with the tag (Page 5, paragraph [0062]; Page 7, paragraph [0078]).
Seitz does not teach that there is an additional RT primer, wherein the additional RT primer comprises in 5' to 3' order: (a) a first generic primer region having a nucleotide sequence that is not complementary to a sequence of the target RNA, (b) a first unique molecular identifier (UMI-A) region, and (c) a RT primer region that is complementary to the sequence located at the 3' end region of the target RNA.
Vollmers, in the same field of endeavor, teaches on using an RT primer that comprises, in 5’ to 3’ order, a first primer region not complementary to the target RNA, a first UID (e.g. UMI), and a RT primer region complementary to a sequence located in the 3’ end region of a target RNA (Page 13463, column 2, paragraph 2; Figure S1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the primers of Seitz with an RT primer of Vollmers. Since both Seitz and Vollmers are in the same field of endeavor (e.g. methods of reverse transcription), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because using unique labels allows identification of molecules from the same RNA molecule after downstream applications live grouping reads after sequencing (Vollmers, Page 13463, column 2, paragraph 2). Furthermore, the RT primer region allows for hybridization with the target and the first primer region allows for later amplification of the target (Vollmers, Page 13463, column 2, paragraph 2; Figure S1).
Claims 12-13 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Seitz (US 20170342409 A1), as applied to claims 1 and 5-7 above, in view of Frey (US 20030113754 A1).
Regarding instant claim 12, Seitz teaches a method of (a) generating a DNA/RNA duplex according to the method of claim 1.
Seitz does not teach generating a double-stranded cDNA molecule comprising the steps of: (b) treating the DNA/RNA duplex with RNase thereby removing the RNA; and (c) incubating an adapter primer comprising a region that is complementary to the sequence located at the 3' end region of the DNA under conditions such that a complementary DNA strand is formed thereby generating a double-stranded cDNA molecule.
Frey, in the same field of endeavor, teaches generating a double-stranded cDNA molecule comprising the steps of: (b) treating the DNA/RNA duplex with RNase thereby removing the RNA (Page 4, paragraph [0074]); and (c) incubating an adapter primer comprising a region that is complementary to the sequence located at the 3' end region of the DNA under conditions such that a complementary DNA strand is formed thereby generating a double-stranded cDNA molecule (Page 4, paragraph [0067]; Page 4, paragraph [0074]). It is noted by the Examiner that while it is not explicitly stated that the primer has a region complementary to the 3’ end region of the DNA, it would necessarily need to be complementary for hybridization to the first strand to then generate a double stranded cDNA as described. Therefore, the cited passages of Frey do teach this limitation.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the first strand cDNA synthesis of Seitz with the second strand synthesis of Frey. Since both Seitz and Frey are in the same field of endeavor (e.g. methods of reverse transcription), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because this would amount to combining prior art elements according to known methods to yield predictable results (see MPEP 2141(III)). Reverse transcription, including first strand cDNA and second strand cDNA synthesis, is a very well-known method in the art.
Regarding instant claim 13, Seitz, in view of Frey, teaches the method of claim 12. Frey further teaches wherein the RNase is RNase-H (Page 4, paragraph [0074]).
Regarding instant claim 15, Seitz, in view of Frey, teaches the method of claim 12. Frey further teaches wherein the complementary DNA strand is formed via a DNA polymerase (Page 4, paragraph [0074]).
Claims 14 is rejected under 35 U.S.C. 103 as being unpatentable over Seitz (US 20170342409 A1) and Frey (US 20030113754 A1), as applied to claims 12-13 and 15 above, and further in view of Vollmers (Genetic measurement of memory B-cell recall using antibody repertoire sequencing, PNAS, July 2013, 1120, 13463-13468).
Regarding instant claim 14, Seitz, in view of Frey, teaches the method of claim 12.
Neither Seitz, nor Frey, teaches wherein the adapter primer, further comprises on the 5' end in 5' to 3' order:(a) a region complementary to a second generic primer having a nucleotide sequence that is not complementary to a sequence of the cDNA, and (b) a region complementary to a second unique molecular identifier (UMI-B).
Vollmers, in the same field of endeavor, teaches on using a second-strand synthesis primer that comprises, in 5’ to 3’ order, a second primer region not complementary to the target cDNA, and a second UID (e.g. UMI; Page 13463, column 2, paragraph 2; Figure S1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the primers of Seitz with an RT primer of Vollmers. Since both Seitz and Vollmers are in the same field of endeavor (e.g. methods of reverse transcription), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because using unique labels allows identification of molecules from the same RNA molecule after downstream applications, like grouping reads after sequencing (Vollmers, Page 13463, column 2, paragraph 2). Furthermore, the second primer region allows for later amplification of the target (Vollmers, Page 13463, column 2, paragraph 2; Figure S1).
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Seitz (US 20170342409 A1) and Frey (US 20030113754 A1), as applied to claims 12-13 and 15 above, and further in view of Vollmers (Genetic measurement of memory B-cell recall using antibody repertoire sequencing, PNAS, July 2013, 1120, 13463-13468) and Karst (Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing, bioRxiv, January 2020, 1-40, https://www.biorxiv.org/content/10.1101/645903v3.full#sec-57).
Regarding instant claim 32, Seitz, in view of Frey, teaches a method of (a) generating a double-stranded cDNA molecule according to the method of claim 12.
Neither Seitz, nor Frey, teaches detecting and removing an artificially recombined DNA molecule (chimera) resulting from PCR-jumping comprising (b) amplifying the double-stranded cDNA molecule via a polymerase chain reaction using a first primer and a second primer that are complementary to the first generic primer region and the second generic primer region, respectively; (c) sequencing the amplified double-stranded cDNA molecule; (d) detecting the artificially recombined DNA molecule which does not have both UMI-A and UMI-B on the same double-stranded cDNA molecule; and (e) removing the artificially recombined DNA molecule in silico.
Vollmers, in the same field of endeavor, teaches on using an RT primer that comprises, in 5’ to 3’ order, a first primer region not complementary to the target RNA, a first UID (e.g. UMI), and a RT primer region complementary to a sequence located in the 3’ end region of a target RNA (Page 13463, column 2, paragraph 2; Figure S1). Vollmers also teaches on using a second-strand synthesis primer that comprises, in 5’ to 3’ order, a second primer region not complementary to the target cDNA, a second UID, and primer region complementary to a sequence located in the 3’ end region of a first strand cDNA (e.g. UMI; Page 13463, column 2, paragraph 2; Figure S1). Finally, Vollmers teaches on PCR amplifying a cDNA using the primer regions not complementary to the target, and further sequencing the cDNA (Page 13463, column 2, paragraph 2; Figure S1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the primers of Seitz, in view of Frey, with the primers of Vollmers. Since Seitz, Frey, and Vollmers are in the same field of endeavor (e.g. methods of reverse transcription), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because using unique labels allows identification of molecules from the same RNA molecule after downstream applications, like grouping reads after sequencing (Vollmers, Page 13463, column 2, paragraph 2). Furthermore, the target complementary primer regions allow for hybridization with the target and the noncomplementary primer regions allow for later amplification of the target (Vollmers, Page 13463, column 2, paragraph 2; Figure S1).
None of the cited references teach using the UMIs for detection and downstream removal of artificially recombined DNA (chimera) in silico.
Karst, in a reasonably pertinent field, teaches on PCR addition of UMIs to each end of a target molecule using primers with 5’ synthetic priming sites, the UMIs in the middle, and 3’ target-specific sequences (Page 3 to Page 5, paragraph 1; Figure 1). Two cycles of PCR add these primers to the target, before subsequent amplification using a first and second primer complementary to the synthetic priming sites (Page 3 to Page 5, paragraph 1; Figure 1). After amplification, the target molecules are sequenced, and undergo data processing to eliminate chimeras from the pool of sequenced targets based on the presence of proper UMI pairs (Page 3 to Page 5, paragraph 1; Figure 1).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Seitz, in view of Frey and Vollmers, with the method of Karst. Since Karst teaches adding UMIs to target nucleic acid molecules for downstream applications, which is reasonably pertinent to the method of Seitz, in view of Frey and Vollmers, one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because using the method and filtering of Karst almost eliminates PCR chimeras, which can otherwise make up 20% of the amplicons. This improves the accuracy of long-read sequencing (Karst, Page 4, lines 98-101 to Page 5, lines 103-104).
Conclusion
All claims are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Allison E Schloop whose telephone number is (703)756-4597. The examiner can normally be reached Monday-Friday 8:30-5 ET.
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/ALLISON E SCHLOOP/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683