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 .
Election/Restrictions
Applicant’s election of the particular species that is ‘extracting the nucleic acid without high temperature or phenol-chloroform extraction’ in the reply filed on 06/19/2026 is acknowledged. In light of the Examiner’s search and consideration of the elected species, the requirement as set forth in the papers of 04/21/2026 is withdrawn.
Objection to the Drawings
Color Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color.
See Figs. 1, 2, 5, 6A and 6B of the Drawings, and the Description of figure 1 ata para 0020 of the Specification as filed.
Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Rejections - 35 USC § 112 - Indefiniteness
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 1-20 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 1-20 are unclear over recitation of the limitations “at least about 1 microgram” and “at least about 1x10-8 per base” (as recited in claim 1), the limitation “pH is about 12.5” (claim 5) and the limitation “about 125 ng” (claim 14). The term “about” adds variability to the recited values, but it is unclear what amount of variability is intended to be encompassed in each case. Neither the claims, the specification as filed, nor the related art provide any clear guidance on, for example, what mass of amplification product is considered to be “at least about 1 microgram”; there is no clear indication as to what amount would be a cutoff to no longer be considered “at least about 1 microgram”. So, for example with the limitation “at least about 1 microgram” it is unclear of 0.5 micrograms would be considered to meet the limitations of the claims.
The limitation “a sequencing floor of at least about 1x10-8 per base” (as recited in claim 1 from which claims 2-20 depend) is further unclear where the term “at least” indicates that values that are greater than 1x10-8 are included in the limitation, but the concept of a “a sequencing floor” would appear to set a minimum of the level of a mutation rate. A “sequencing resolution floor” represents the baseline error rate of a sequencing technology, which acts as a noise threshold, so it is unclear if the claims intended to encompass mutation rates that are higher than 1x10-8.
Claim 12 is unclear over recitation of the limitation “the rolling circle amplification comprises a buffer having an EDTA concentration of 1 uM” because it is unclear if the limitation is intended to address some buffer that is added to the amplification reaction (e.g.: a storage buffer for an enzyme with an EDTA concentration of 1 uM in the storage buffer), or if the limitation is intended to require that the rolling circle amplification is perfumed in a solution in which the final DTA concentration is 1 uM.
Claim 13 is unclear over recitation of the limitation “the rolling circle amplification lacks a primer annealing step” because rolling circle amplification (RCA) is a method known in the art where a primer hybridized to a circular template is extended by a polymerase. It is unclear how a process of RCA can be performed without a primer annealing to the template.
Claim Rejections - 35 USC § 112 – Written Description
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 1-20 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 written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The instant rejection is appropriate where the claims are directed to methods that generically encompass any methods of fragmenting and amplifying any nucleic acids in combination with the requirements that “the method produces at least about 1 microgram of amplification products at a mutation rate with a sequencing resolution floor of at least about 1x10-8 per base.” While it is noted that these limitations have been addressed earlier in this Office Action under 35 USC 112 as unclear, the claims are addressed here in the interests of customer service and compact prosecution.
Because the claims generically encompass a variety of different methodological steps, whiles requiring a particular outcome of the steps with regard to the amount of amplification product and a mutation rate of the product, it is relevant to address the fact that different variables encompassed by the breadth of the claims will alter the outcome of the results of rolling circle amplification with regard to both product quantity and quality.
The related art demonstrates that a number of factors may affect the yield of RCA product. For example, Inoue et al teaches that the amount of template can alter the amount of RCA products (e.g.: Fig 6). This is relevant where the claims require a particular amount of product (i.e.: at least about 1 microgram), but the claims are generic with regard to any particular template amount. In this regard the specification teaches that the claim may encompass the use of as little as 10ng of template (e.g.: para 0059), but exemplifies only the use of 125ng of nucleic acid to routinely produce at least 1 microgram of product (e.g.: para 0082). Similarly, while the specification exemplifies the use of phi29 polymerase (e.g.: para 0083), the claims generically encompass the use of any polymerase in performing RCA. But the related art of Tang et al (2016) teaches that even using the same templates, different polymerases provide markedly different amounts of RCA products (e.g.: Figs 2, 3, 4).
Furthermore it is noted that the claims generically encompass any isolated nucleic acids, and the specification provides that the subject nucleic acids may be DNA or RNA (e.g.: para 0040). But the specification exemplifies only the analysis of gDNA (e.g.: para 0080). The breadth of the encompassed isolated nucleic acids of the claims and the requirement for the recited mutation rate is relevant in light of the requirements for RCA of RNA templates which would include either reverse transcription of isolated RNA fragments, or use of an RNA-dependent RNA polymerase for transcription-based RCA of RNA fragments. The related art of Gout et al (2017) (cited on the IDS of 01/02/2026) teaches that reverse transcriptases and RNA polymerases have error rates that would preclude making products of the required mutation rates of the claims.
The claims generically encompass any fragmenting methods, and the use of any enzyme that may fragment the isolated nucleic acids. The specification asserts that the in embodiments of the methods “sonication and/or other fragmentases may be used as MNase alternatives” (para 0080). But the specification teaches that sonication results in elevated mutation rates (e.g.: para 0090). And the prior art teaches that the high energy from a sonication step used for nucleic acid fragmentation u may result in DNA damage (e.g.: Wang et al (2016)), and that fragmentation of nucleic acids using different enzymes may result in the formation of sequencing artifacts (e.g.: Gregory et al (2020)).
Finally it is noted that even in the particular exemplification of the methods of the disclosure, the results do not clearly indicate that “a mutation rate with a sequencing resolution floor of at least about 1x10-8 per base” is achieved (it is noted that this limitations has been addressed earlier in this Office Action as unclear). Table 1 of the specification teaches mutation rate estimates as base pair substitutions (bps)/base, and provides that the smallest measured bps/base (provided by ‘Hiseq2500 MMR-’) is 1.22 x 10-7.
When considering the variability encompassed by the different elements of the claimed methods, the skilled artisan, given the teachings of the application as filed and the related art, can not select from the myriad of different possible particular methodological steps the combination of particular elements that will provide the required functionality as required by the claims. As such the rejection of claims as encompassing subject matter which was not adequately described by the application as filed is appropriate.
Claim Rejections - 35 USC § 102
It is noted that several claim limitations have been addressed earlier in this Office Action under 35 USC 112 as unclear. In the interests of customer service and compact prosecution the practical steps of the claims in view of the prior art are addressed here.
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.
Claim(s) 1, 7, 9, 11, 13-16 and 18-20 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lou et al (2013) as cited on the IDS of 01/10/2026 including the Supporting Information.
Relevant to the limitations of claim 1, Lou et al teaches a Circle Sequencing method including fragmenting isolated nucleic acid, denaturing the fragments, circularizing fragments, and amplifying the circularized fragments (e.g.: Supporting Information – Procedures A, B, C and D.). The methodological steps of the prior art meet the broad functional limitations of producing at least about 1 microgram of product, and a mutation rate with a sequencing resolution floor of at least about 1x10-8 per base.
Relevant to claim 7, Lou et al the analysis of RCA products that are produced without the use of DNA repair enzymes (e.g.: Supporting Information – Procedures A, B and C).
Relevant to claim 9, Lou et al teaches kinase treatment of fragments, and digestion of non-circularized fragments (e.g.: Supporting Information – Procedures B and C).
Relevant to claim 11, Lou et al teaches RCA primed with random primers (e.g.: Supporting Information – Procedure D).
Relevant to claim 13, Lou et al teaches that RCA can be performed without DNA repair enzymes (e.g.: Figure 2A) (i.e.: the limitation “lacks a primer annealing step and/or DNA repair enzymes” is interpreted to mean that the claim may be directed to a method that lack either one of the recited elements).
Relevant to claims 14-16, Lou et al teaches isolated nucleic acid that meets the broadly recited limitation of claim 14 (e.g.: Supporting Information – Procedure A) and is gDNA form a subject or microorganism (claims 15 and 16) (e.g.: p.19877 – Circle Sequencing).
Relevant to claims 18-20, Lou et al teaches sequencing RCA products, including steps of amplification of adaptor-ligated products, and detection of mutations (e.g.: Supporting Information – Procedure E; p.4, variant calling).
Claim Rejections - 35 USC § 103
It is noted that several claim limitations have been addressed earlier in this Office Action under 35 USC 112 as unclear. In the interests of customer service and compact prosecution the practical steps of the claims in view of the prior art are addressed here.
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.
Claim(s) 1, 7, 9-11, 13-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lou et al (2013) as cited on the IDS of 01/10/2026 including the Supporting Information.
Lou et al teaches the methods of 1, 7, 9, 11, 13-16 and 18-20, as detailed above in this Office Action.
Lou et al does not exemplify the analysis of nucleic acid fragments that are less that 150 base pairs (relevant to the instant rejection of claim 10), or DNA obtained from a tissue suspected of being diseased (relevant to the instant rejection of claim 17).
Relevant to the instant rejection of claim 10, Lou et al teaches that the average size of the fragments should be slightly less than 1/3 of the average read length of the sequencing used (e.g.: Supporting Information – Procedure A).
Relevant to the instant rejection of claim 17, Lou et al teaches that the methods of Circle Sequencing are applicable to detect of variation in complex samples such as tumors.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have created fragments less than 150 base pairs for circularization. Lou et al teaches that fragment size is important, and related to the read length of the sequencing technology that is used using, such that fragment length ensures least three repeats of each sequence in one read length. Lou et al thus provides that fragment length is a result effective variable, and thus it would have been obvious to the skilled artisan to optimize fragment length, including a length of less than 150 bases, for any particular sequencing technology. Even for the same sequencing technology exemplified by Lou et al, the skilled artisan may be motivated to use smaller fragment to have more repeats of each sequence in one read length, thus increasing the ability to subtract errors as taught by Step 4B in Figure 1.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to performed the methods of Lou et al using a tumor tissue. The skilled artisan would have been motivated to analyze tumor tissue based on the expressed teachings of Lou et al that the methods of Circle Sequencing are suitable for use in contexts where rare genetic variants are sought, such as in the analysis of mutations in genetically heterogeneous tumors.
Claim(s) 1, 2, 6-11, 13-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lou et al (2013) as cited on the IDS of 01/10/2026 including the Supporting Information in view of Fritsch et al (2018) and Wang et al (2016).
Lou et al teaches the methods of 1, 7, 9, 11, 13-16 and 18-20, and renders obvious the methods of claims 10 and 17, as detailed above in this Office Action.
Lou et al does not provide for fragmenting nucleic acids using an enzyme (relevant to the instant rejection of claim 2), circularization at 25oC (relevant to the instant rejection of 6), or methods where fragments are not size selected (relevant to the instant rejection of 8). However, such methods in the context of sequence analysis were known in the prior art.
Fritsch et al teaches sequence analysis comprising RCA of circularized fragmented nucleic acids. Relevant to the instant rejection of claims 2 and 8, Fritsch et al teaches the fragmentation of sample nucleic acids using an enzyme, and the further use of the fragments with only a clean-up procedure without any size selection (e.g.: p.3 - RNase III Fragmentation and RNA Clean Up). Relevant to t the instant rejection of claim 6, Fritsch et al exemplifies the circularization of nucleic acid fragments at 25oC (e.g.: p.3 - RNA Circularization and Rolling Circle Reverse Transcription).
Further relevant to the instant rejection of claims, Wang et al teaches the detection of mutations in nucleic acid samples using an RCA based analysis of nucleic acid fragments (e.g.: Figure 1). Wang et al teaches that fragmentation of nucleic acids by sonication can introduce damage to the nucleic acid that appears as mutations in the sequencing analysis, and that such sonication-induced mutation may be prevented with alternative methods of fragmentation such as using enzyme digestion instead of sonication to fragment the sample nucleic acid (e.g.: p.3).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have used the methodological steps of enzymatic digestion of a nucleic acid sample (as taught by Fritsch et al and Wang et al), as well as the circularization of fragments at 25oC and lack or a size selection step (each taught by Fritsch et al) in the Circle Sequencing of Lou et al. The skilled artisan would have been motivated to use an enzymatic digestion of fragments based on the expressed teachings of Fritsch et al that an enzymatic digestion is suitable for creating a library of nucleic acids that can be circularized without a size selection step (thus making for a more efficient method of creating circularized nucleic acid fragments), and the teaching of Wang et al that use of an enzyme to fragment nucleic acids prevents mutations formed by sonication thus allowing for a more accurate detection of true variants in the sample nucleic acid. Additionally, using the fragmentation and ligation methods of Fritsch et al (e.g.: ligation at 25oC) in the methods of Lou et al would have been the simple substitution of one known prior art element for another with predictable results.
Claim(s) 3 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lou et al (2013) as cited on the IDS of 01/10/2026 including the Supporting Information in view of Fritsch et al (2018) and Wang et al (2016), as applied to claims 1, 2, 6-11, 13-20 above, and further in view of Allan et al (2012).
Lou et al including the Supporting Information in view of Fritsch et al and Wang et renders obvious method of nucleic acid sample analysis using Circle Sequencing and fragmentation to the nucleic acid using a fragmenting enzyme.
Lou et al including the Supporting Information in view of Fritsch et al and Wang et does not specifically suggest using micrococcal nuclease (MNase) as a fragmenting enzyme. However, the use of MNase to fragment genomic DNA for subsequent sequence analysis was known in the prior art.
Allan et al teaches fragmentation of genomic DNA using MNase (e.g.: p.162 – Nucleosomal DNA preparation).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have performed the methods rendered obvious by Lou et al including the Supporting Information in view of Fritsch et al and Wang et using MNase (as taught by Allan et al) to fragment a nucleic acid sample, for example the gDNA of Lou et al. The skilled artisan would have been motivated to use MNase based on the expressed teachings of Allan et al that S. cerevisiae DNA can be digested to 147bp (further relevant to the rejection of claim 10) fragments using MNase, and the teachings of Lou et al regarding the length of fragments suitable for sequence analysis using Circle Sequencing. The skilled artisan would have had a reasonable expectation of success based on the teachings of Allan et al which provide for the efficient digestion of DNA using MNase.
Claim(s) 1, 4, 5, 7, 9, 11, 13-16 and 18-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lou et al (2013) as cited on the IDS of 01/10/2026 including the Supporting Information in view of Ehrlich et al (1958) and Bruskov et al (2002).
Lou et al including the Supporting Information teaches the methods of 1, 7, 9, 11, 13-16 and 18-20, as detailed above in this Office Action.
Lou et al does not exemplify denaturing nucleic acid with alkaline pH (claim 4) of about pH 12.5 (claim 5) . However, alkaline denaturation of nucleic acid was known in the prior art.
Ehrlich et al provides for the alkaline denaturation of double stranded nucleic acid, and teaches that DNA undergoes a substantial reduction in specific viscosity, indicative of a separation of the two polynucleotide chains that make up the native DNA molecule, under alkaline conditions in the pH range of 11.5 to 12.0 and that the denaturation is complete and irreversible above pH 12.0.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have used the methodological step of alkaline denaturation of a nucleic acid sample (as taught by Ehrlich et al) in the Circle Sequencing of Lou et al. Where Lou et al teaches the denaturation of nucleic acids, the skilled artisan would have been motivated to use an alkaline digestion step based on the expressed teachings of Erlich et al that such methods allow for the complete denaturation of nucleic acids, and the expressed teachings of Bruskov et al that heat can induce formation of mutations. Where Lou et al includes teachings related to methods of reducing mutations caused during sample preparation, it would have been obvious to the skilled artisan to substitute the known methods of the prior art in order to avoid formation of mutations.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Sakhabutdinova et al (2017) teaches the use of T4 RNA ligase in the circularization of DNA to create templates for RCA. Hadi et al (2020) teaches that a typical yield in RCA is approximately 27 µg of amplified DNA per 100 µl reaction.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHEN THOMAS KAPUSHOC whose telephone number is (571)272-3312. The examiner can normally be reached M-F, 8am-5pm.
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Stephen Kapushoc
Primary Examiner
Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683
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