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 the Application
The Reply filed December 22, 2025 is acknowledged.
Claims 1-11 are pending and are being examined on the merits.
Response to Arguments
Applicant’s arguments filed December 22, 2025 have been fully considered.
The following objections and rejections are WITHDRAWN in view of Applicant’s arguments and claim amendment:
Objections to claims 1 and 9
Rejection of claims 10-11 under 35 USC § 112(d)
The following rejections are MODIFIED:
Rejection of claims 1-11 under 35 USC § 112(a), lack of enablement
Response to arguments regarding lack of enablement rejections
Applicant argues that the lack of enablement rejections should be withdrawn because instant claim 1 has been amended to clarify the scope of the invention (Remarks, p. 5). Applicant also argues that Fig. 2 provides sufficient description for the claimed methods, as does the instant specification. For example, paras. 31-36 of the specification describe performing PCR with a particular number of cycles on the retrieved RCA product using a primer pair, and teaches that doing so results in a PCR product with P1 and P2 adapters which is then circularized (Remarks, p. 7).
The Examiner disagrees. First, the method described in instant claim 1 does not comprise the embodiment described in Fig. 21, and even if it did, the method described in Fig. 2 is not presumed to be enabled. Second, as to the teachings of the specification, although there is some overlap between the method described in the specification and the claim 1 method, there are also significant differences between in the methods. For example, the specification teaches using P1 and P2 adapters in the circularization step, but there are no adapters recited in the claim 1 method.
These arguments are not persuasive. The rejections are modified in view of the instant claim amendments.
Claim Interpretation
Claim 1 recites a series of steps, including steps (a) through (e) which are directed to, in part, hybridizing a “first oligonucleotide” (e.g., a padlock probe) to a target RNA or cDNA comprised within a tissue, and amplifying the padlock probe with rolling circle amplification (RCA). The “primary rolonies” (i.e., the RCA product) are then removed from the tissue. These steps are depicted in instant Fig. 2, as follows:
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As shown above, the RNA or cDNA strand comprises the “target sequence”, and the padlock probe comprises the “region of interest” (ROI), which hybridizes to the “target sequence” (target). Thus, the ROI is complementary to the target. Further, since the padlock probe is serving as the template for amplification, the primary rolonies will comprise the target/complement of the ROI (ROI’).
Claim 1, steps (f) and (g)(i) then require fragmenting the primary rolonies into a plurality of “second oligonucleotides” (which are single-stranded and linear), and hybridizing first PCR primers (P1) at the 3’ ends of the second oligonucleotides, and then amplifying the second oligonucleotides, thus creating “multiplied second oligonucleotides”, as follows (“FS”: flanking sequence):
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Claim 1, step (g)(ii) then requires hybridizing a second PCR primer at the 5’ ends of the multiplied second oligonucleotides, thereby obtaining “third oligonucleotides”, as follows:
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Thus, the “third oligonucleotide” is a partially single-stranded, partially double-stranded construct comprising P1, FS1’, Target’/ROI, FS2’ on the “bottom” strand, with P2 (which also comprises P1’) creating the “top” strand.
Claim 1, step (g)(ii) states that the third oligonucleotide comprises the first PCR primer, the target sequence and the second PCR primer, that the first and second PCR primers hybridize to regions flanking the target sequence. Finally, steps (h) and (i) require that these third oligonucleotides are multiplied by PCR, and the circularized by ligating together the first and second PCR primer.
Regarding step (g)(ii), while the third oligonucleotide does comprise the first and second
PCR primer, it does not comprise the target (rather, it comprises target’). In addition, the first and second PCR primers do not flank either the target (because it’s not there) or target’. Rather, P1 is on the 5’ end of the third oligonucleotide and P2 is hybridized to P1.
Regarding steps (h) and (i), it is not clear how such a structure could be PCR amplified, nor is it clear how the first and second primers could be ligated together to form a circular construct.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-11 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
In considering whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue,” the factors set forth in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) should be considered. These factors are:
The breadth of the claims,
The nature of the invention,
The state of the prior art,
The level of one of ordinary skill in the art,
The level of predictability in the art,
The amount of direction provided by the inventor,
The existence of working examples; and
The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Each of these will be discussed in turn below.
Nature of the Invention and the Breadth of the Claims
The invention of instant claim 1 is directed to a method for sequencing a target nucleic acid derived from a tissue, comprising, in part, hybridizing a padlock probe to the target sequence, amplifying the padlock probe with rolling circle amplification (RCA), fragmenting the RCA concatemers into linear segments, amplifying the linear segments, re-circularizing the linear segments, and finally, performing additional RCA followed by sequencing the amplicons.
The dependent claims further define additional features of the various oligonucleotide structures used and the fragmentation, ligation and amplification steps. In view of the disclosure in the specification, claims 1-11 are not overly broad.
The State of the Prior Art
The prior art generally teaches methods for detecting target nucleic acids in situ using padlock probes, followed by multiple rounds of RCA and then sequencing the RCA products (see discussion of Ke, Dahl and Daugharthy teachings in prior art section below). However, the prior art does not teach, at least, generating the instant partially double-stranded “third nucleotide” structure following fragmentation and amplification of an RCA product, nor does it teach amplifying such a structure to create multiple “third nucleotide” structures, or circularizing such amplified structures.
Level of One of Ordinary Skill in the Art
One of ordinary skill in the art would have the knowledge equivalent to a graduate-level degree in molecular biology.
Level of Predictability and Amount of Direction Provided by Inventor
While the level of predictability in the art of assays involving amplification techniques, including RCA, is generally high, the specification does not provide any teaching relating to amplifying the “third nucleotide” structures to create multiple “third nucleotide” structures, or circularizing such amplified structures. In particular, it does not teach how to perform PCR using a second primer that is hybridized to the 5’ end of a template, and which is hence “pointing” in the wrong direction.
Existence of Working Examples
The specification provides one working example describing detecting target nucleic acids in situ using padlock probes, followed by multiple rounds of RCA and then sequencing the RCA products (paras. 42-44). However, the example does not describe in detail the particular step of amplifying the “third nucleotide” structures to create multiple “third nucleotide” structures or the step of circularizing such structures; rather it simply states that PCR is performed using P1 and P2 primers. However, as shown above, in the claim 1 method, at least, the P2 primer is not in the correct orientation to perform PCR.
Quantity of Experimentation Needed to Make or Use the Invention Based on the Content of the Disclosure
Considering the disclosures in the specification as a whole, to make and use the invention of instant claim 1, one of ordinary skill in the art would have to determine how to perform amplification of the “third nucleotide” structures to create multiple “third nucleotide” structures and how to circularize these structures. In addition, the ordinary artisan would have to determine how to perform PCR using a second primer that is “pointing” in the wrong direction. If the teachings in the specification provide no more than a “plan” for those of skill in the art to experiment using the technology, the entire breadth of the claims are not enabled. MPEP 2164.06(b).
Claims 2-11 depend from claim 1, and consequently incorporate the lack of enablement issues in claim 1.
For the above reasons, one of ordinary skill in the art would not be able to make and use the claimed invention without undue experimentation.
Appropriate correction is required.
Prior Art
As noted above, the prior art does not teach, at least, generating the instant “third nucleotide” structure following fragmentation of an RCA product, nor does it teach amplifying such a structure to create multiple “third nucleotide” structures.
The closest prior art is Ke2 (In situ sequencing for RNA analysis in preserved tissue and cells, Nature Methods, 10(9): 857-860, 2013), Dahl3 (Circle-to-circle amplification for precise and sensitive DNA analysis, PNAS, 101(13): 4548-4553, 2004) and Daugharthy4 (WO 2020/076976 A1).
Ke teaches a method with the steps of hybridizing a padlock probe comprising a barcode to a cDNA sequence in situ in a tissue section, followed by RCA and sequencing of the amplification product (Fig. 1).
Dahl teaches circle-to-circle amplification (C2CA), where an RCA product is fragmented into linear pieces, followed by re-circularizing the linear pieces, and a subsequent round of RCA (Fig. 1).
Daugharthy teaches a method with the steps of hybridizing a padlock probe comprising a barcode to a cDNA sequence in situ in a tissue section, followed by RCA. The amplification products are then removed from the matrix and sequenced in vitro.
Conclusion
Claims 1-11 are being examined and are rejected. 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 CAROLYN GREENE whose telephone number is (571)272-3240. The examiner can normally be reached M-Th 7:30-5:30 EST.
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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.
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/CAROLYN L GREENE/Primary Examiner, Art Unit 1681
1 For example, Fig. 2 depicts P1 and P2 in an orientation which is understood in the art to represent tailed primers. However, claim 1 does not require tailed primers. Fig. 2 also depicts that P1 and P2 appear on the same strand of the single-stranded amplification product, which is recited in claim 1 step (g)(ii), however, for the reasons discussed below, the prior steps of claim 1 will not generate such a product. Further, claim 1, step(g)(ii) generates a partially-double stranded molecule, also discussed below, whereas no such molecule appears in Fig. 2, and so on.
2 Ke was cited in the PTO-892 Notice of References Cited mailed October 1, 2025.
3 Dahl was cited in the PTO-892 Notice of References Cited mailed October 1, 2025.
4 Daugharthy was cited in the PTO-892 Notice of References Cited mailed October 1, 2025.
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