Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-13 and 16-22 are pending and under investigation. Claims 7, 11, and 20 have been amended. Claims 21 and 22 are new. Claims 1, 3, and 12 are independent claims.
Response to Arguments
Objections Withdrawn
The objection to claim 7 is withdrawn following the applicants’ amendments.
Rejections Withdrawn
The rejection of claims 1-4, and 12-13 under 35 U.S.C. 112(a) has been withdrawn following the applicants’ amendments.
The rejection of claims 1-13 and 16-20 under 35 U.S.C. 102(a)(1)/102(a)(2) as being anticipated by Zheng et al. is withdrawn.
Rejections Maintained
In regards to claims the rejection of 1-13 and 16-20 under 35. U.S.C. 112(b), the applicant's arguments filed 08/20/2025 have been fully considered but they are not persuasive. The applicant argument “given that there are only four types of nucleotides, some degree of nucleotide overlap is inherently unavoidable” does not overcome the rejection. While applicant acknowledges that overlap may occur, applicant fails to provide any objective standard, quantitative threshold, or functional criterion by which one of ordinary skill in the art could determine when a nucleic acid sequence is “partially the same” as another sequence. Applicants’ acknowledgment that “some degree of overlap is inherently unavoidable” further demonstrates that the claim lacks a clear boundary, as the claim provides no guidance as to what degree of overlap is required or sufficient. Furthermore, as “overlap is inherently unavoidable”, the lack of guidance would suggest that this doesn’t provide any limitation at all. As such, the claim fails to inform, with reasonable certainty, the scope of the claimed invention, as required by 35 U.S.C. 112(b). Accordingly, the indefiniteness rejection is maintained.
As stated in the previous office action, the applicants should consider amending the claims to specify a defined level of sequence identity or number of consecutive base pairs in common that would result in the claimed stem-loop structure formation.
New Rejections
Claim Rejections - 35 USC § 112
Claims 21-22 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.
Claims 21 and 22 depend on claims 1 and 12, respectively, which are rejected for indefiniteness above. Claims 21 and 22 do not fix the deficiency and are therefore indefinite as well.
Claim Interpretation
Claim 1 states “a 3'-end sequence of the first tag primer is partially or completely the same as the first universal sequencing sequence of the reverse primer at the 5'-end, the first tag primer further comprises a first tag sequence, and a 3'-end sequence of the second universal primer is partially or completely the same as the second universal sequencing sequence of the nested primer at the 5'-end.” the same sequence encompasses as few as one nucleotide. So the “ends” have at least one nucleotide the same.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (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.
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 1-13 and 16-22 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng (WO 2019/023924 A1, of record), in view of Sugino et al. (US 2014/0315211 A1, published Oct. 23, 2014).
In regards to claims 1, 3 and 12, Zheng teaches a method for preparing sequencing libraries using nested amplification for enrichment of target nucleic acid sequences, including a first amplification using primers specific to a target region (see Figs. 1-3, [8], [113], and throughout), purification of amplification products prior to further amplification (see [19], [106], [110], and throughout), a second amplification using nested primers and universal adaptor-related primers to generate sequencing ready libraries (See Fig. 1-3, [8], [113], and throughout), and use of universal adaptor sequences and universal primers for library construction (see [121]-[135] and throughout). Thus, Zheng teaches the overall nested PCR workflow, purification between amplification rounds and use of universal primer sequences to enable sequencing library preparation. Zheng does not explicitly disclose incorporating a universal sequencing sequence at the 5’-ends of specific primers in the precise primer architecture recited, nor designing primers such that terminal primer sequences are “partially or completely the same” as terminal sequences of other primers used in subsequent amplification rounds.
However, Sugino teaches PCR methods in which primers or adaptors are designed with terminal sequences that are identical or substantially identical to other primer/adaptor sequences such that the terminal sequence can anneal to each other or to complementary primer regions and teaches that the sequence identity relationships between primer/adaptor ends are intentionally engineered to control downstream amplification behavior and specificity (see Abstract, Figs. 1,3-8, 11, 13, and 15, and throughout). Therefore, Sugino teaches designing PCR primers/adaptors in which terminal sequences are identical or substantially identical to other primer sequences utilized in later amplification steps.
Zheng seeks to improve efficiency and specificity of nester PCR-based sequencing library preparations, while Sugino teaches that designing primers/adaptors with identical or substantially identical terminal sequences is an effective and predictable technique for controlling PCR amplification behavior and improving specificity. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to utilize primers with terminal sequences which can anneal to each other or complementary primer regions as taught by Sugino in the multiplex nested PCR workflow taught by Zheng. One would be motivated to modify Zheng’s nested PCR primers to incorporate universal sequencing sequences at primer 5’ ends and to design subsequent primers whose terminal regions are partially or completely the same as those universal sequences as taught by Sugino, in order to improve primer compatibility across amplification rounds, facilitate efficient universal primer amplification, enhance specificity and control of nested amplification and simply sequencing library construction. Such a modification merely applies Sugino’s known suppression-PCR terminal-sequence design principles to Zheng’s nested sequencing-library workflow to achieve predictable improvements.
Zheng already teaches nested amplification with universal adaptor related primers, and Sugino teaches that primer terminal sequences can be intentionally designed to be identical or substantially identical to other primer/adaptor sequences to control PCR behavior. Because both references operate in the same technical field of PCR primer design and sequencing library preparation, a person of ordinary skill in the art would have had a reasonable expectation of success in combining these teachings. The modification requires only routine primer sequence design and does not alter the fundamental amplification chemistry taught by Zheng.
Thus the combination of Zheng and Sugino render obvious using a reverse primer comprising a specific 3’ region and universal sequencing sequence at the 5’ end, using a nested primer comprising a 3’ region and second universal sequencing sequence at the 5’ end, and using tag primers and universal primers whose terminal regions are partially or completely the same as the universal sequencing sequences as recited in Claim 1.
In regards to claims 2, 4, and 5, Zheng teaches the use of tag(index) primers for multiplex sequencing library preparation (see Fig. 3, [129], [131], [133], [232]) and Sugino teaches designing primers having terminal sequences that are identical or substantially identical to other primer/adaptors. It would have been obvious to one of ordinary skill in the art to configure the second universal primer of the claim 1 as a tag primer comprising a tag sequence while maintaining terminal sequence identity with the universal sequencing sequence of the nested primer, as recited in Claim 2, in order to enable multiplex sample identification while preserving primer compatibility across amplification rounds. Such modification represent a predictable application of known primer tagging techniques and would have been made with a reasonable expectation of success.
In regards to claim 6, 11, 17 and 20-22, Zheng teaches the use of UMI barcodes with random sequence of least about 5 (such as at least about any one of 10, 15, 20, or 25) randomly and/or degenerately designed nucleobases (see [129]) and the use of molecular tags of “at least about 13 nucleotides” (see [9], [80]-[83], [86]-[88], and throughout). One of ordinary skill in the art would recognize that the length of the molecular tag or barcode used would be adjustable and optimizable for the assay being run, and would recognize that longer a tag is, the more samples it could be used to distinguish between. Furthermore, which primer these tags are incorporated into would be a design choice obvious to one of ordinary skill in the art.
In regards to claims 7-8, and 18, Zheng teaches a multiplexed approach to enrich multiple target nucleic acids, wherein the forward and reverse primers and nested primers are in a primer pool composed of a plurality of primers respectively targeting different targeted regions ([84]-[86], and throughout). Zheng further teaches that these regions may adjacent and/or overlap (see [137]).
In regards to claim 9 and 19, Zheng teaches using primers that amplify in opposite directions, and details that these primers contain distinct 5’ tags to reduce or eliminate PCR amplification products form primers in opposite directions (see [88], [139]).
In regards to claim 10, Zheng teaches that first and second rounds of amplification are performed for at least 2 amplification cycles and provides multiple examples including about 5-30 cycles in paragraph 103 (see [86], [93],[102]-[105]) reading on the limitation of 2 to 30 cycles of PCR amplification.
In regards to claims 12-13, and 16-20, Zheng teaches methods of preparing high-throughput sequencing library based on nested multiplex PCR, and teaches all the limitations of the kits claimed, implying that the a they were in possession and in use of the contents of the kit at the time of filing as outlined in the rejection above.
Conclusion
No claim is allowed
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/MATTHEW HAROLD RAYMONDA/ Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681