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 .
Election/Restrictions
Applicant’s election of Group III (claims 9-11, 13, 15, 17, 18, and 20) in the reply filed on 8/21/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Applicants’ analysis of the limitations of elected claim 9 and non-elected claim 22 (p.8 of the Remarks of 08/21/2026) are noted. The propriety of any election requirement will be reconsidered throughout prosecution and examination of the elected invention.
Claims 1-4, 8-11, 13, 15, 17-18, 20-24, 26, 28, 30-31, and 33 are pending.
Claims 1-4, 8, 21-24, 26, 28, 30-31, and 33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 8/21/2026.
Claims 9-11, 13, 15, 17, 18, and 20 are being examined on the merits.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement (e.g., see paragraphs [0063 and 0066]). 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The disclosure is objected to because of the following informalities: The specification contains references to colors in the Drawings (for example, paragraphs [0046-0047 and 0126]). The Drawings provided are in black and white. Therefore, references to color in the figures should be avoided where possible.
Appropriate correction is required.
The use of the terms “Bioruptor” and “SYBR” (paragraph [0046]), which are trade names or marks used in commerce, have been noted in this application. These terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The examples above are not an exhaustive list of unmarked trade names or marks used in commerce throughout the specification. Please carefully read through and properly notate each instance.
Claim Objections
Claims 15 and 17 are objected to because of the following informalities:
Claim 15 reads “the ratio of the target DNA over the linear DNA being greater than 1000” and should read reads “the ratio of the target circular DNA over the linear DNA [[being]]is greater than 1000” to maintain consistent claim terminology and proper grammar.
Claim 17 reads “wherein the ratio of the target DNA over the linear DNA” and should read “wherein the ratio of the target circular DNA over the linear DNA” to maintain consistent claim terminology.
Appropriate correction is required.
Claim Rejections - 35 USC § 112b - 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 9-11, 13, 15, 17, 18, and 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.
Claim 9 is directed to a method for “enriching and quantifying” extrachromosomal circular DNA. There are active steps related to the enriching of eccDNA but there are no steps of quantifying the eccDNA. The only quantification performed is on the added plasmids or mtDNA and chromosomal DNA (step (iii)). It is unclear how the aim of the method can be achieved when only the quality control values are assessed in the method of claim 9. According to the specification, quantifying the respective amounts of the added plasmids and/or quantifying the amounts of mtDNA and chromosomal DNA is a method of quality control to ensure that circular DNA has been enriched over linear DNA but is not a quantification of eccDNA (paragraphs [0115-0117, 0128]).
Claims 10-11, 13, 15, 17, 18, and 20 depend from claim 9, inherit this deficiency, and are rejected on the same basis.
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.
Claims 9-11, 15, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Møller (Møller et al., Nature Communications 2018) in view of Alanin (Alanin et al., Plasmid, May 2021).
Claim 9: Møller teaches a method of enriching and quantifying extrachromosomal circular DNA (eccDNA) in tissues (pg 2, col 1, paragraph 4; Methods – Extrachromosomal circular DNA enrichment for Circle-Seq; Methods – Quantification of eccDNA based on internal controls). Møller teaches that the eccDNA comprises mitochondrial DNA and non-organelle eccDNA (“Our data further reveal that large parts of the human genome can be found on eccDNA”, pg 8, col 1, paragraph 2; Methods – Removal of linear and mitochondrial DNA for Circle-Seq). Møller teaches lysing the tissue and treating the tissue with a proteinase (concurrently) and then spiking the sample with a mixture of plasmids of different sizes (Methods - Cell lysis for Circle-Seq). Møller then teaches treating with an exonuclease which digests linear polynucleotides (“Remaining linear DNA was removed by exonuclease”, Methods – Removal of linear and mitochondrial DNA for Circle-Seq). Møller teaches recovery of the eccDNA and quantification of amounts of the spiked-in circular plasmids (Figure 2C).
While Møller teaches adding proteinase K concurrently with lysing the cells rather than after spiking the sample with the control plasmids, with respect to the order of steps, it is noted that the courts have held that any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C. Therefore, the claimed order of steps is an obvious variant of the steps of the cited prior art.
Møller teaches that the inclusion of circular plasmids in known amounts are internal controls for measuring the sensitivity of purification/detection of circular DNA (pg 4, col 1, paragraph 2). Møller also teaches determining the efficiency of linear DNA digestion by quantifying an amount of a linear endogenous locus from chromosomal DNA (“We confirmed that linear DNA was completely removed after exonuclease treatment using quantitative PCR (qPCR) on a gene absent from eccDNA as a marker (COX5B, Supplementary Fig. 1a, b)”, pg 2, col 2, paragraph 2).
Møller does not teach spiking in a first plasmid that is linearized and quantifying the amount. However, use of linearized plasmids as spiked-in, internal controls for monitoring effectiveness of exonuclease digestion is known in the art, as taught by Alanin.
Alanin teaches spiking in linearized pKJK5-GFP to samples containing circular plasmids of known sizes (Figure 1). Alanin teaches quantifying the amount of linearized plasmid DNA after exonuclease treatment and sequencing (Figure 2A) as well as quantifying the amount of the circular DNA plasmids (Figure 3A).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Møller with that of Alanin to include a linearized plasmid as linear DNA control. One would be motivated to do so given the assertion by Alanin that including a known amount of a linearized plasmid allows for assessment of the efficiency of exonuclease treatment in relation to the total sequencing reads of the population of plasmids (circular and otherwise, 3.1 Efficiency of linear DNA degradation by exonuclease treatment). One would have a reasonable expectation of success given that Alanin is performing this measurement on circular and linear DNA in the process of degrading linear DNA to enrich for circular DNA via treatment with an exonuclease, a similar process as that being employed by Møller.
Claim 10: The methodology of Møller utilizes rolling circle amplification (Figure 1a). However, Alanin teaches that use of amplification disproportionately enriches the sample for smaller-sized plasmids and that skipping this step results in higher coverage of bigger plasmids (3.3. Omitting amplification and using the direct metamobilome approach is more sensitive to relatively large circular elements than including amplification before sequencing). One would be motivated to skip the amplification step given that Møller teaches detection of larger plasmids (26.3kb, Figure 2c) but that the coverage of said plasmids is much lower than the smaller plasmids given the amplification bias of phi29 (pg 8, col 2, paragraph 3). One would have a reasonable expectation of success given that Alanin shows that circular DNA can be effectively enriched and quantified without an amplification step (Figure 3). Møller teaches quantifying the nucleic acid amount via next-generation sequencing (Methods – Uniquely mapped eccDNA pipeline for Circle-Seq data), as does Alanin (2.4. Sequencing library; Figure 2 and 3).
Claim 11: Møller teaches eccDNA comprises eccDNA at least 10kb and between 0.5-10kb (Figure 2a and b).
Claim 15 and 17: Møller teaches quantifying a target circular DNA (mtDNA) and a linear DNA (chromosomal DNA). Møller teaches measuring remaining linear DNA by amplifying the COX5B locus and a mtDNA locus via qPCR (Methods – Quantification of genomes per sample). Møller does not explicitly teach calculating a ratio, but it would be obvious to one of skill in the art that measurement of a linear DNA vs. measurement of circular DNA following exonuclease treatment provides an estimate of the amount of linear DNA left following exonuclease digestion and thus provides information on the enrichment of circular DNA (mtDNA) following digestion. Furthermore, while Møller does not teach that the linear DNA is a locus on chromosome 17, the use of chromosome 17 rather than chromosome 2 (with the COX5B locus) is obvious substitution that would yield similar results of enabling the determination of remaining linear DNA in a sample following exonuclease digestion. Additionally, Møller does not explicitly teach that the enrichment of the target circular DNA is greater than 1000, however Møller does teach that COX5B is completely removed following exonuclease treatment, as confirmed by qPCR (pg 2, col 2, paragraph 2). Therefore, with a denominator of zero (or near zero, see Supplementary figure 1a, b), the enrichment of circular DNA over linear DNA is necessarily near infinite (greater than 1000).
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Møller (Møller et al., Nature Communications 2018) in view of Alanin (Alanin et al., Plasmid, May 2021) as applied to claims 9-11, 15, and 17 above, and further in view of Kumar (Kumar et al., Molecular Cancer Research 2017).
The teachings of Møller in view of Alanin are detailed above. Relevant to the instantly rejected claim, Møller teaches that eccDNA is found in many types of cancers (pg 2, col 1, paragraph 2).
Møller in view of Alanin do not teach enriching and quantifying eccDNA in two or more tissues wherein the first is a tumor tissue and the second is a normal tissue obtained adjacent to the tumor tissue. However, enrichment and quantification of eccDNA from tumor and adjacent normal tissue is known in the art, as taught by Kumar.
Kumar teaches enrichment and quantification of eccDNA in lung cancer patients using lung cancer tissue samples and adjoining normal lung tissue (pg 5, paragraph 3, Figure S2A-C).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Møller in view of Alanin to quantify the amount of eccDNA from paired tumor and adjacent normal tissues as taught by Kumar. One would be motivated to do so given the assertion by Kumar that there are differences in eccDNA derived from tumor tissue as opposed to those derived from normal tissues which may allow for monitoring of successful tumor eradication post-surgery (pg 7, paragraph 2). One would have a reasonable expectation of success given that Møller also performs eccDNA enrichment and purification on tissue samples from humans.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Møller (Møller et al., Nature Communications 2018) in view of Alanin (Alanin et al., Plasmid, May 2021) as applied to claims 9-11, 15, and 17 above, and further in view of AddGene (pCMV-Cre (plasmid #123133, 2019)) and CLONTECH Laboratories, Inc. (pEGFP-C1, 1997).
The teachings of Møller in view of Alanin are detailed above. Relevant to the instantly rejected claim, Møller teaches quantifying a target circular DNA (mtDNA) and a linear DNA (chromosomal DNA). Møller teaches measuring remaining linear DNA by amplifying the COX5B locus and a mtDNA locus via qPCR (Methods – Quantification of genomes per sample). Møller does not explicitly teach calculating a ratio, but it would be obvious to one of skill in the art that measurement of a linear DNA vs. measurement of circular DNA following exonuclease treatment provides an estimate of the amount of linear DNA left following exonuclease digestion and thus provides information on the enrichment of circular DNA (mtDNA) following digestion. In additiona Alanin teaches performing a similar quantification of both circular DNA (plasmids, e.g., pBR322) and linear DNA (linearized plasmid, pKJK5GFP) following exonuclease digestion and sequencing as a readout of efficient degradation of linear DNA in the sample via exonuclease treatment (Figures 2 and 3).
Møller in view of Alanin does not teach that the linearized plasmid DNA is pEGFP-C1 or that the circular plasmid DNA is pCMV-Cre. However, it would be a simple substitution, with predictable results, to perform the same methodology on commercially available plasmids such as pEGFP-C1 (CLONTECH) and pCMV-Cre (AddGene) which have known sequences and restriction sites for linearization.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Møller (Møller et al., Nature Communications 2018) in view of Alanin (Alanin et al., Plasmid, May 2021) as applied to claims 9-11, 15, and 17 above, and further in view of Møller 2020 (Møller et al., DNA Electrophoresis 2020; cited on IDS of 4/30/2024), Qiagen (HiSpeed Plasmid Purification Handbook, 2012), Schuster (Schuster et al., US 20190127729 A1), Zieler (US 20150329853 A1), and Rounseville (Rounseville et al., US 20160265036 A1).
The teachings of Møller in view of Alanin are detailed above. Relevant to the instantly rejected claim, in view of Alanin teaches in step (i) lysing the tissue by incubating the tissue in a lysis buffer (Methods – Cell lysis for Circle-Seq, Møller) but does not specify the components of said lysis buffer. Møller in view of Alanin teaches in step (ii) treating the plasmid-spiked sample with an alkaline buffer (Methods – Extrachromosomal circular DNA enrichment for Circle-Seq, Møller) but does not teach adding RNase. Møller in view of Alanin teaches that treatment with the proteinase comprises adding proteinase K and teaches incubating at 50º C but does not teach only incubating the sample for about 2 hours (Methods – Cell lysis for Circle-Seq, Møller). Møller in view of Alanin teaches alkaline treating the samples but does not specify that the alkaline buffere comprises NaOH and SDS or that sample is incubated for 5 minutes (Methods – Extrachromosomal circular DNA enrichment for Circle-Seq, Møller). Møller in view of Alanin teaches recovering the eccDNA from a buffer exchange column post-exonuclease treatment (2.3. Removal of genomic DNA, Alanin).
In summary, Møller in view of Alanin do not teach:
that the lysis buffer comprises Tris-HCl, EDTA, NaCl, and SDS;
that the incubation with proteinase K at 50º C occurs for about 2 hours;
that the plasmid-spiked sample is treated with RNase and incubated at about 50º C for about 1 hour; or
that the alkaline treatment comprises adding an alkaline buffer containing NaOH and SDS and incubating for about 5 minutes.
However, each of these features as they relate to isolation of DNA from biological samples is known in the art, as taught by Møller 2020, Qiagen, Schuster, Zieler, and Rounseville.
The lysis buffer:
Schuster teaches that is known in the art that animal tissue can be lysed by a lysis buffer comprising SDS, NaCl, EDTA, and Tris-HCl (paragraph [0005]). Møller 2020 teaches lysis of tissue samples by lysis buffer comprising Tris-Cl and KCl (another commonly used salt) and describes including SDS in the lysis buffer as well (2.1 Cell Lysis and Note 7). The Qiagen Plasmid Midi kit teaches Tris-Cl, EDTA, and SDS in the lysis/resuspension buffer (Buffer P1 combined with Buffer P2 for alkaline lysis, pg 15-16 (steps 4 and 5) and pg 28).
Proteinase K treatment at 50º C for 2 hours:
Zieler teaches lysing cells by the addition of proteinase K to a sample and incubating said sample at 50º C for 2 hours (paragraph [0062]).
Treating the plasmid-spiked sample with RNase and incubating for 1 hour at 50º C:
Rounseville teaches performing digestion by adding RNase A to the sample and incubating for 1 hour at 50º C (paragraph [0127]).
Alkaline buffer containing NaOH and SDS with incubation for 5 minutes:
Qiagen teaches adding an alkaline buffer (Buffer P2) containing NaOH and SDS and allowing the sample to incubate at room temperate for 5 minutes (pg 16, step 5, and pg 28).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Møller in view of Alanin to use art disclosed variations of lysis, RNase A treatment, and alkaline buffer compositions as taught by Møller 2020, Qiagen, Schuster, Zieler, and Rounseville. Regarding alterations of incubation times and buffer components, Rounseville teaches that “One of ordinary skill in the art can vary the reaction conditions, such as time, temperature, amount of nuclease, salt concentration, metal ion concentration, or other factors in order to optimize sensitivity and/or specificity of the methods” (paragraph [0127]). Additionally, 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. Therefore, absent any unexpected results, the claimed specifications of buffer compositions, temperatures, and times of incubation are obvious over the cited prior art. One would have a reasonable expectation of success given the assertion by Rounseville that optimization of sensitivity/specificity is a known aspect of sample treatment in the art.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683
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