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 Response and the Request for Continued Examination, each filed May 12, 2026, are acknowledged.
Claims 49-75, 77 and 79-80 were pending. Claims 49-73, 75, 77 and 79-80 are being examined on the merits. Claim 74 is canceled.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on May 12, 2026 has been entered.
Response to Arguments
Applicant’s arguments filed May 12, 2026 have been fully considered.
The following rejections are WITHDRAWN in view of Applicant’s arguments and amendments to the claims:
Rejection of claim 74 under 35 USC § 112(b), indefiniteness
The following rejections are MODIFIED in view of Applicant’s arguments and amendments to the claims:
Prior art rejections
Response to arguments regarding prior art rejections
The prior art rejections are modified in view of the instant claim amendments. To the extent that Applicant’s arguments are relevant to the modified rejections, the Examiner notes the following.
Applicant argues that the prior art rejections should be withdrawn because the prior art does not teach or suggest all of the limitations of independent claims 49 and 77 for several reasons (Remarks, p. 9).
First, Applicant argues that Cao does not teach a genus of methyltransferases and the statement in Cao at para. 27 that “a methyltransferase, such as DNMT1, may require conditions, such as buffer conditions, that do not include ions …” should be interpreted to mean that Cao was only referring to DNMT1 (Remarks, pp. 9-10). Applicant additionally notes that Cao states “in embodiments where the primer extension step and the methylation step are carried out in the same vessel and media, one needn’t purify the DNA since a chelating agent is used to chelate magnesium to provide a magnesium free buffer for the methyl-transfer reaction” (Remarks, p. 10).
The Examiner disagrees. According to the Merriam-Webster dictionary, the phrase “such as” is “used to introduce an example or series of examples”. Thus, clearly Cao is referring to a group of methyltransferases that may require certain conditions, with DNMT1 being an example of such a methyltransferase in that group of methyltransferases. Further, “may” is “used to indicate possibility or probability”. Thus, interpreting that statement in Cao to be limited to DNMT1, or to indicate that all methyltransferase enzymes require buffer conditions that do not include ions, contravenes the basic English usage of “such as” and “may”. As to the second point, regarding the teachings in Cao as to the use of a chelating agent, the Examiner notes that Cao refers to such a step as being limited to “a certain embodiment”, and thus is not applicable to all embodiments. Further, the ordinary artisan understands that if the methyltransferase used is not inhibited by and/or requires the presence of magnesium cations in solution, then one would not chelate the magnesium cations prior to the methyltransferase step. Further, the instant specification states (p. 79):
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Thus, the instant specification expressly admits that the ordinary artisan has the knowledge and skill to select appropriate concentrations of cations in certain nucleic acid assay solutions. Consequently, the ordinary artisan would be capable of optimizing cation concentrations in a modified Cao assay.
Second, Applicant states:
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(Remarks, p. 10).
It isn’t clear what Applicant is arguing here. Cao teaches the use of a polymerase which is magnesium dependent in the amplification step, followed by an embodiment of a methylation step using a methyltransferase that is inhibited by magnesium. Cao further teaches adding the chelating step in between the amplification step and the methylation step. Thus, in such an embodiment, magnesium would be present in the amplification step, as required by the polymerase, and absent in the methylation step, as required by that particular embodiment of the methyltransferase. Consequently, it is not clear what would be “kill[ed]” by this chelation step. Further, such an embodiment is not relevant to the rejection of record.
Third, Applicant argues that independent claims 49 and 77 have been amended to require that the instant method does not include a DNA purifying step, in contrast to Cao’s one-pot method which requires either DNA purification or alternating between chelating and replenishing Mg2+ (Remarks, p. 11).
The Examiner disagrees. As noted above, Cao teaches multiple embodiments of methods comprising amplification and methylation steps, not all of which are relevant to the instantly claimed invention. Further, claims 49 and 77 stand rejected over a combination of Cao together with the teachings of secondary references. Applicant has not addressed that combination of teachings here.
Fourth, Applicant argues that while Cao discloses a number of methyltransferases, that collection of methyltransferases does not constitute a genus (Remarks, p. 11).
Applicant has not provided any reasoning for why the group of methyltransferases taught by Cao would not be considered a genus according to the MPEP, and thus, the Examiner cannot further address this argument.
Fifth, Applicant argues that Cao recites a specific embodiment using DNMT1, and although Cao recites other methyltransferases, those other methyltransferases may not be substituted for DNMT1 because they would produce inoperable embodiments, and in particular, the substitution of DNMT1 with DNMT3a, DNMT3b, Dam, DRM2 and CMT3 would result in inoperable embodiments (Remarks, pp. 11-12).
The Examiner disagrees and directs Applicant’s attention to MPEP 2121(I) which states that “[w]hen the reference relied on expressly anticipates or makes obvious all of the elements of the claimed invention, the reference is presumed to be operable”, and also MPEP 2121.01(II) which states that “’a non-enabling reference may qualify as prior art for the purpose of determining obviousness’”. Finally, the Examiner notes that the prior art rejections do not cite substituting DNMT1 with DNMT3a, DNMT3b, Dam, DRM2 and CMT3, so these arguments are not relevant to the rejection of record.
Sixth, Applicant disagrees with the statements in the prior art rejections as to detecting low quantities of DNA (Remarks, pp. 12-13).
The prior art rejections below have been modified to clarify this issue.
Seventh, Applicant disagrees that DNMT5 is known in the art to be useful for performing methylation functions in assays similar to Cao’s (Remarks, p. 12).
The Examiner disagrees. Cao teaches that their assay, at least in part, is directed to treating hemi-methylated double stranded templates with methyl transferase to methylate cytosine on the newly synthesized stand locations where the original double stranded template was methylated (para. 10). DNMT5 is known for having such a capability. Further, the prior art rejections below have been modified to clarify this issue.
Finally, as to the dependent claims, Applicant argues that the additional secondary references do not cure the deficiencies of the art cited in the rejections of the independent claims (Remarks, pp. 14-15).
The Examiner disagrees with Applicant’s characterization of the teachings of the references cited against the independent claims, and thus disagrees with this position as well.
These arguments are not persuasive. The prior rejections are modified in view of the instant claim amendments.
Information Disclosure Statement
The Information Disclosure Statements submitted June 3, 2026 and June 5, 2026 have been considered.
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.
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 49-60, 63-72, 75, 77 and 79-80 are rejected under 35 U.S.C. 103 as being unpatentable over Cao1 (US Patent App. Pub. No. 2020/0063213 A1) in view of Dumesic2 (ATP Hydrolysis by the SNF2 Domain of Dnmt5 is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA, Molecular Cell, 79(1): 127-139, 2020) and Siedentop (Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions, Catalysts, 11(10): 1183, 1-24, 2021).
Regarding independent claim 49 and dependent claims 50-53, 56-58, 63-64 and 67-68,
Cao teaches …
A method of amplifying methylated DNA, comprising: (a) denaturing double stranded methylated DNA (dsDNA) at a first temperature and lowering the first temperature to a second temperature to copy a target sequence on one strand of the denatured double stranded DNA (dsDNA) with a DNA polymerase, optionally a strand displacing DNA polymerase, optionally Bst DNA polymerase, and at least one target specific DNA oligonucleotide primer (Fig. 1, 2nd p.; paras. 25, 43, 47, 54, 127: denature at 94°C, and lower to second temperature to copy target sequence, e.g., 64°C);
wherein the dsDNA is symmetrically methylated and the copying produces a hemimethylated dsDNA substrate (Fig. 1, 2nd p.: after the primer extension step);
and (b) lowering the second temperature to a third temperature and contacting the hemimethylated dsDNA substrate with a methyltransferase enzyme comprising methyltransferase activity, optionally further comprising a SAM methyl donor group (paras. 59, 101: lower temperature to 37°C and incubate with methyltransferase enzyme, 104; Fig. 1, 2nd p.: DNMT1 incubation step);
wherein the thermal cycling amplification comprising steps (a) and (b) is repeated at least once in the same reaction vessel, optionally between 2-50 times (paras. 10, 14: 2-4 times, 25: 1 to 3 … 2 to 6, 43);
and wherein the method produces an amplified methylated dsDNA product, which is optionally symmetrically methylated (Fig. 1, 2nd p.: after the DNMT1 incubation step).
Regarding the limitation requiring that the hemimethylated dsDNA substrate is contacted with an enzyme comprising methyltransferase activity and ATP hydrolysis (ATPase) activity, Cao teaches a genus of methyltransferases, which are used to treat “hemi-methylated double stranded DNA … to produce methylated double stranded DNA fragments which results in replication of the methylation status … of the original template” (para. 25), and specifically teaches DNMT1 (para. 25). DNMT1 comprises methyltransferase activity, but does not additionally comprise ATPase activity. However, Dumesic teaches another DNMT enzyme, specifically DNMT5, and teaches that DNMT5 comprises both methyltransferase and SNF2 ATPase domains and corresponding activity, and further that the additional ATPase activity contributes to higher specificity for hemimethylated DNA compared to DNMT1. Dumesic additionally teaches that DNMT5 has > 1000-fold greater activity for hemimethylated DNA compared to unmethylated DNA (Fig. 1; abstract; p. 128, left col., paras. 2-3; p. 131, left col., para. 2; p. 135, right col., para. 2; p. 136, right col., para. 2). Dumesic additionally teaches that DNMT5 has hemimethylation activity at 23°C (p. e2: DNA methyltransferase assay).
Regarding the limitations requiring that the cation concentration is sufficient to support both DNA polymerase activity and ATPase activity of the methyltransferase enzyme, and that the DNA is not purified between steps, Cao does not explicitly teach a specific embodiment where magnesium ions are present in both steps of the reaction or that the same buffer is used in both steps. However, Cao does teach carrying out the amplification and methylation steps in the same vessel, and that, in some embodiments “a methyl-transferase … may require conditions, that do not include … cations … which may be a component of a PCR reaction” (para. 27). Thus, the ordinary artisan would understand from “may require” that some methyltransferases require a lack of cations while others do not. The ordinary artisan would also understand that if both the polymerase and methyltransferase being used require magnesium ions, then there would be no reason to purify the DNA between steps. Further,
Cao teaches 3.67 mM MgSO4 3 (para. 103) for DNA polymerase activity, while Dumesic teaches using 1mM MgCl2 for DNMT5 methyltransferase activity (p. e2: DNA methyltransferase assay). It is generally understood in the art, and taught in Siedentop (e.g., abstract), that when a reaction requires the use of multiple, perhaps two, enzymes, the components in the reaction mix should be optimized so to maximize the activity of both enzymes. Siedentop also teaches strategies for optimizing cation concentrations in such situations (sections 2, 3, 3.1.3). Thus, since Cao and Dumesic teach magnesium concentrations appropriate for the polymerase and DNMT5, respectively, and since Siedentop teaches the need to optimize magnesium concentrations and strategies for doing so, the ordinary artisan would have optimized the magnesium concentration, using the prior art conditions as a guide, to arrive at a cation concentration that is sufficient to support both DNA polymerase activity and ATPase activity of the methyltransferase enzyme, and would not have added an unnecessary purification DNA step between the amplification and methylations steps.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Cao method to incorporate the DNMT5 enzyme of Dumesic. The ordinary artisan would have been motivated to incorporate the DNMT5 enzyme into the Cao method with the expectation that doing so would result in the advantage of an assay with an improved efficiency for generating a fully methylated double stranded product, as DNMT5 has higher specificity for hemimethylated DNA as compared to the Cao DNMT1 enzyme. It would have been additionally obvious to use the methyltransferase enzyme at a temperature which is known in the art to be an active temperature for the enzyme. The ordinary artisan would further have optimized the temperature through routine experimentation to customize the assay as desired. The ordinary artisan would have had an expectation of success as Cao teaches using various enzymes, and because DNMT5 is known in the art to be useful for performing methylation functions in assays with hemimethylated DNA substrates.
It would have been further obvious to modify the Cao method to incorporate magnesium ions in both steps of the reaction and to not purify the DNA between steps, as Cao teaches that polymerases require magnesium, as do at least some methyltransferases, and Dumesic teaches a DNMT reaction mix comprising magnesium ions. Further, Siedentop teaches that a reaction mix should be optimized to maximize the activity of both enzymes used in the assay, and provides guidance for doing so. Thus, the ordinary artisan would have been motivated to optimize the buffers and buffer components through routine experimentation to customize the assay as desired, including to use a single buffer for both steps of the assay, as doing so would result in the advantage of a lower cost assay. The ordinary artisan would have had an expectation of success as the design and modification of nucleic acid assay reaction components is well-known in the art, as taught by Siedentop.
Regarding dependent claim 54, Cao additionally teaches further determining the presence of methylated DNA in the amplified methylated dsDNA product (para. 43).
Regarding dependent claims 55 and 75, Cao teaches further adding sequencing adapters to the amplified methylated dsDNA product for sequencing (para. 54: Illumina adapters), as recited in claim 55, and that after a final thermal amplification cycle comprising steps (a) and (b) is completed, the method further comprises the step of sequencing the amplified methylated dsDNA product and/or subjecting the amplified methylated dsDNA product to epigenetic analysis (paras. 25, 54), as recited in claim 75.
Regarding dependent claims 59-60, Cao teaches that the polymerase step, excludes the addition of a chelating agent, and specifically EDTA (para. 27). Cao also teaches carrying out the amplification and methylation steps in the same vessel, and that, in some embodiments, “a methyl-transferase … may require conditions, that do not include … cations … which may be a component of a PCR reaction” (para. 27). Thus, the ordinary artisan would understand from “may require” that some methyltransferases require a lack of cations while others do not, and would further understand that, in embodiments that do not require a lack of cations, a chelating agent would not be added to the reaction volume.
Regarding dependent claims 65-66 and 69, Cao additionally teaches that the target specific primer comprises a tag at the 5’ and/or 3’ end (para. 123), as recited in claim 65, and that the tag comprises biotin (para. 123), as recited in claim 66. Cao additionally teaches that the biotin-tagged amplicons are isolated/immobilized using an avidin binding partner attached to a (solid) substrate (para. 123), as recited in claim 69.
Regarding dependent claims 70-71, Cao and Dumesic additionally teach that the enzyme in (b) has de novo methylation activity less than that of DNMT1, as recited in claim 70, and optionally no detectable de novo methylation activity, as recited in claim 71. Specifically, Cao teaches that DNMT1 has de novo and maintenance methylation activities (paras. 26, 114), and Dumesic teaches that DNMT5 has maintenance methylation activity only, but no de novo methylation activity (p. 128, left col., para. 2).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further modify the modified Cao method, discussed above, to incorporate a methyltransferase enzyme with no de novo methylation activity. The ordinary artisan would have been motivated to incorporate the recited enzyme into the Cao method with the expectation that doing so would result in the advantage of an assay with an improved efficiency for generating a fully methylated double stranded product, as the recited enzyme has higher specificity for hemimethylated DNA as compared to the Cao DNMT1 enzyme. MPEP 2144.07 states that “[t]he selection of a known material based on its suitability for its intended use support[s] a prima facie obvious determination”. The ordinary artisan would have had an expectation of success as Cao teaches using various enzymes, including the genus of enzymes comprising the DNMT enzymes, and because DNMT5 is known in the art to be useful for performing methylation functions in assays with hemimethylated DNA substrates.
Regarding dependent claim 72, Dumesic does not teach that the enzyme in (b) has the property of being thermofunctional at a temperature of up to about 30°C. However, Dumesic does teach that the enzyme is thermofunctional for at least part of the recited range, i.e., up to at least 23°C (p. e2: DNA methyltransferase assay performed at 23°C).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further modify the modified Cao method, discussed above, to optimize the choice of enzymes and their working temperatures. The ordinary artisan would have been motivated to do so to customize the assay as needed through routine optimization to select an enzyme with the desired properties, and then to use that enzyme at a temperature consistent with its activity profile. The ordinary artisan would also have had an expectation of success in doing so as designing and modifying nucleic acid assay reaction components to select appropriate enzymes is well-known in the art.
Regarding dependent claims 79-80, Cao additionally teaches that first temperature is between about 90°C to about 100°C (para. 127: denature at 94°C) and the second temperature is between about 50°C to about 80°C (para. 127: amplify at, e.g., 64°C), as recited in claim 79. Cao additionally teaches that first temperature is between about 92°C to about 98°C (para. 127: denature at 94°C) and the second temperature is between about 55°C to about 72°C (para. 127: amplify at, e.g., 64°C), as recited in claim 80.
Regarding independent claim 77, Cao teaches …
A method of determining the methylation status of a target sequence in a biological fluid, comprising amplifying methylated double-stranded DNA from the biological fluid to produce an amplified symmetrically methylated dsDNA product, the method comprising: (a) denaturing double stranded methylated DNA (dsDNA) at a first temperature and lowering the first temperature to a second temperature to copy a target sequence on one strand of the denatured double stranded DNA (dsDNA) from the biological fluid with a DNA polymerase, and at least one target specific DNA oligonucleotide primer (Fig. 1, 2nd p.; paras. 25, 31, 43, 47, 54, 56, 127: denature at 94°C, and lower to second temperature to copy target sequence, e.g., 64°C, 145);
wherein the dsDNA is symmetrically methylated and the copying produces a hemimethylated dsDNA substrate (Fig. 1, 2nd p.: after the primer extension step);
and (b) lowering the second temperature to a third temperature and contacting the hemimethylated dsDNA substrate with a methyltransferase enzyme comprising methyltransferase activity (paras. 59, 101: lower temperature to 37°C and incubate with methyltransferase enzyme, 104; Fig. 1, 2nd p.: DNMT1 incubation step);
wherein the thermal cycling amplification comprising steps (a) and (b) is repeated at least once in the same reaction vessel (paras. 10, 14: 2-4 times, 25: 1 to 3 … 2 to 6, 43);
and wherein the method produces an amplified symmetrically methylated dsDNA product (Fig. 1, 2nd p.: after the DNMT1 incubation step), and wherein the methylation status is associated with a medical condition (paras. 17, 23-24, 145).
Regarding the limitation requiring that the hemimethylated dsDNA substrate is contacted with an enzyme comprising methyltransferase activity and ATP hydrolysis (ATPase) activity, Cao teaches a genus of methyltransferases, which are used to treat “hemi-methylated double stranded DNA … to produce methylated double stranded DNA fragments which results in replication of the methylation status … of the original template” (para. 25), and specifically teaches DNMT1 (para. 25). DNMT1 comprises methyltransferase activity, but does not additionally comprise ATPase activity. However, Dumesic teaches another DNMT enzyme, specifically DNMT5, and teaches that DNMT5 comprises both methyltransferase and SNF2 ATPase domains and corresponding activity, and further that the additional ATPase activity contributes to higher specificity for hemimethylated DNA compared to DNMT1. Dumesic additionally teaches that DNMT5 has > 1000-fold greater activity for hemimethylated DNA compared to unmethylated DNA (Fig. 1; abstract; p. 128, left col., paras. 2-3; p. 131, left col., para. 2; p. 135, right col., para. 2; p. 136, right col., para. 2). Dumesic additionally teaches that DNMT5 has hemimethylation activity at 23°C (p. e2: DNA methyltransferase assay).
Regarding the limitations requiring that the cation concentration is sufficient to support both DNA polymerase activity and ATPase activity of the methyltransferase enzyme, and that the DNA is not purified between steps, Cao does not explicitly teach a specific embodiment where magnesium ions are present in both steps of the reaction or that the same buffer is used in both steps. However, Cao does teach carrying out the amplification and methylation steps in the same vessel, and that, in some embodiments “a methyl-transferase … may require conditions, that do not include … cations … which may be a component of a PCR reaction” (para. 27). Thus, the ordinary artisan would understand from “may require” that some methyltransferases require a lack of cations while others do not. The ordinary artisan would also understand that if both the polymerase and methyltransferase being used require magnesium ions, then there would be no reason to purify the DNA between steps. Further,
Cao teaches 3.67 mM MgSO4 4 (para. 103) for DNA polymerase activity, while Dumesic teaches using 1mM MgCl2 for DNMT5 methyltransferase activity (p. e2: DNA methyltransferase assay). It is generally understood in the art, and taught in Siedentop (e.g., abstract), that when a reaction requires the use of multiple, perhaps two, enzymes, the components in the reaction mix should be optimized so to maximize the activity of both enzymes. Siedentop also teaches strategies for optimizing cation concentrations in such situations (sections 2, 3, 3.1.3). Thus, since Cao and Dumesic teach magnesium concentrations appropriate for the polymerase and DNMT5, respectively, and since Siedentop teaches the need to optimize magnesium concentrations and strategies for doing so, the ordinary artisan would have optimized the magnesium concentration, using the prior art conditions as a guide, to arrive at a cation concentration that is sufficient to support both DNA polymerase activity and ATPase activity of the methyltransferase enzyme, and would not have added an unnecessary purification DNA step between the amplification and methylations steps.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Cao method to incorporate the DNMT5 enzyme of Dumesic. The ordinary artisan would have been motivated to incorporate the DNMT5 enzyme into the Cao method with the expectation that doing so would result in the advantage of an assay with an improved efficiency for generating a fully methylated double stranded product, as DNMT5 has higher specificity for hemimethylated DNA as compared to the Cao DNMT1 enzyme. It would have been additionally obvious to use the methyltransferase enzyme at a temperature which is known in the art to be an active temperature for the enzyme. The ordinary artisan would further have optimized the temperature through routine experimentation to customize the assay as desired. The ordinary artisan would have had an expectation of success as Cao teaches using various enzymes, and because DNMT5 is known in the art to be useful for performing methylation functions in assays with hemimethylated DNA substrates.
It would have been further obvious to modify the Cao method to incorporate magnesium ions in both steps of the reaction and to not purify the DNA between steps, as Cao teaches that polymerases require magnesium, as do at least some methyltransferases, and Dumesic teaches a DNMT reaction mix comprising magnesium ions. Further, Siedentop teaches that a reaction mix should be optimized to maximize the activity of both enzymes used in the assay, and provides guidance for doing so. Thus, the ordinary artisan would have been motivated to optimize the buffers and buffer components to through routine experimentation to customize the assay as desired, including to use a single buffer for both steps of the assay, as doing so would result in the advantage of a lower cost assay. The ordinary artisan would have had an expectation of success as the design and modification of nucleic acid assay reaction components is well-known in the art, as taught by Siedentop.
Claims 61-62 are rejected under 35 U.S.C. 103 as being unpatentable over Cao5 (US Patent App. Pub. No. 2020/0063213 A1) in view of Dumesic6 (ATP Hydrolysis by the SNF2 Domain of Dnmt5 is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA, Molecular Cell, 79(1): 127-139, 2020) and Siedentop (Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions, Catalysts, 11(10): 1183, 1-24, 2021) as applied to claim 49 above, and further in view of Gormley7 (US Patent No. 9,868,982).
Regarding dependent claims 61-62, Gormley additionally teaches that the target specific primer further comprises an adapter, which is optionally methylated. Further, Gormley teaches that “[t]he presence of methylated adaptors in these … polynucleotides facilitates treatment of such polynucleotides with bisulfite for the purposes of determining methylation status of cytosine bases in the target portion of the … polynucleotides … the adapters ligated onto the ends of the targets are fully methylated and are, therefore, resistant to bisulfite induced alterations. Thus, any unmethylated cystosine bases present … originate exclusively in the target nucleic acid portion of the polynucleotide” (col. 5, ll. 12-40).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further modify the modified Cao method, discussed above, to incorporate the various Gormley adapters. The ordinary artisan would have been motivated to incorporate these various elements with the expectation that doing so would result in the advantage of an amplified product with improved properties for downstream applications. For example, the methylated adapters and barcodes of Gormley are useful for deconvoluting sequence data. The ordinary artisan would have had an expectation of success as the design and modification of nucleic acid libraries which include various adapters is well-known in the art.
Claim 73 is rejected under 35 U.S.C. 103 as being unpatentable over Cao8 (US Patent App. Pub. No. 2020/0063213 A1) in view of Dumesic9 (ATP Hydrolysis by the SNF2 Domain of Dnmt5 is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA, Molecular Cell, 79(1): 127-139, 2020) and Siedentop (Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions, Catalysts, 11(10): 1183, 1-24, 2021) as applied to claim 49 above, and further in view of Brown10 (WO 2024/256581 A1; effectively filed date June 14, 2023)
Regarding dependent claim 73, Brown teaches SEQ ID NO: 73 (p. 115), which is identical
to instant SEQ ID NO: 1.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further modify the modified Cao method, discussed above, to incorporate the DNMT5, as recited in the Brown sequence. The ordinary artisan would have been motivated to do so to customize the assay as needed through routine optimization to select an enzyme with the desired properties. The ordinary artisan would also have had an expectation of success in doing so as designing and modifying nucleic acid assay reaction components to select appropriate enzymes is well-known in the art.
Conclusion
Claims 49-73, 75, 77 and 79-80 are being examined, and are rejected. No claims are allowed.
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.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
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 Cao was cited in the Information Disclosure Statement submitted October 29, 2025.
2 Dumesic was cited in the Information Disclosure Statement submitted October 29, 2025.
3 0.55 uL of a 100 mM stock solution diluted in a 15 uL reaction volume = final concentration of 3.67 mM.
4 0.55 uL of a 100 mM stock solution diluted in a 15 uL reaction volume = final concentration of 3.67 mM.
5 Cao was cited in the Information Disclosure Statement submitted October 29, 2025.
6 Dumesic was cited in the Information Disclosure Statement submitted October 29, 2025.
7 Gormley was cited in the PTO-892 Notice of References Cited mailed February 25, 2026.
8 Cao was cited in the Information Disclosure Statement submitted October 29, 2025.
9 Dumesic was cited in the Information Disclosure Statement submitted October 29, 2025.
10 Brown was cited in the PTO-892 Notice of References Cited mailed February 25, 2025.
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