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 .
Claims 1-3, 6-8, 10, 12-13, 15-16, 18, 20-23, 25, 63, 69-70, 73, 76, 79 and 85 are pending and are being examined on the merits.
Information Disclosure Statement
The Information Disclosure Statement submitted December 3, 2025 has been considered, except for NPL no. 31, which was not submitted. However, it appears that NPL nos. 5 and 31 may be referring to the same document, as they have similar citations in the IDS and have the same date. NPL no. 5 has been reviewed.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Specifically, a hyperlink appears in para. 308.
Claim Objections
Claims 7-8 ae objected to because of the following informalities:
In claim 7, there are two semicolons at the end of step b). One should be removed.
In claim 8, there are two semicolons at the end of step b). One should be removed.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 8, 10, 12 and 85 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 8 recites the limitation "the treated DNA molecules" in step e). There is insufficient antecedent basis for this limitation in the claim. Claim 8 does not previously recite “treated” DNA molecules.
Claims 10 and 12 depend directly or indirectly from claim 8, and consequently
incorporate the lack of antecedent basis issues of claim 8.
Claim 85 recites “the first strands” and “the second strands” in each of a) and f). There is insufficient antecedent basis for these limitations in the claim. Claim 85 does not previously recited “first” and “second” strands.
Claim Rejections - 35 USC § 102
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 85 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Balasubramanian1 (WO 2022/023753 A1).
Regarding independent claim 85, Balasubramanian teaches …
A kit comprising one or more of: a) a reagent for synthesizing first and second complementary strands (helicase); b) a reagent for glucosylating a 5-hydroxmethylated cytosine (DNA glucosyltransferase); c) a reagent for methylating a cytosine (DNMT1); d) a reagent for deaminating an unmodified cytosine (deaminase) (para. 149).
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.
Claims 1, 3, 6, 20-23, 25, 63, 69-70, 73, 76 and 79 are rejected under 35 U.S.C. 103 as being unpatentable over Balasubramanian (WO 2022/023753 A1) in view of Zhou (US Patent 2021/0404007 A1) and Ahn (Asymmetrical barcode adapter-assisted recovery of duplicate reads and error correction strategy to detect rare mutations in circulating tumor DNA, Scientific Reports, 7:46678, 1-9, 2017).
Regarding independent claim 1 and dependent claims 3, 6, 20-23, 25 and 69-70,
Balasubramanian teaches …
A method of analyzing DNA molecules in a sample, optionally blood, the DNA molecules, optionally cell-free DNA, optionally from a mammal, comprising first and second strands and adapters, which are optionally ligated to the DNA molecules, the method comprising: a) synthesizing first complementary strands which are complementary to the first strands and second complementary strands which are complementary to the second strands (Figs. 1A-B, 2A-B; paras. 88, 92, 123);
b) glucosylating a 5-hydroxymethylated cytosine in at least one first or second strand before or after synthesizing the first and second complementary strands (Figs. 1A-B, 2A-B);
c) methylating a cytosine in at least one first complementary strand or second complementary strand, wherein the methylation converts a hemimethylated CpG to a fully methylated CpG, optionally with a methyltransferase, optionally DNMT1 or DNMT5 (Figs. 1A-B, 2A-B; para. 87);
d) deaminating an unmodified cytosine, optionally with bisulfite conversion, in at least one first or second strand, thereby producing treated DNA molecules (Figs. 1A-B, 2A-B; para. 4);
and e) sequencing at least a portion of the treated DNA molecules (Figs. 1A-B, 2A-B).
Balasubramanian does not teach that the adapters are asymmetric. However, Zhou teaches asymmetric adapters, optionally Y-shaped adapters, optionally with barcodes (paras. 19, 6; Figs. 2, 6B-C). Zhou additionally teaches that each asymmetric adapter comprises a deamination-sensitive cytosine that is an unmethylated cytosine (para. 20).
Further, Ahn teaches that the use of asymmetrical adapters provides advantages in NGS sequencing, including error correction, improved accuracy and sensitivity in variant calling and reduced false positives (e.g., p. 2, Results section; p. 6, para. 3; p. 7, paras. 3-4).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Balasubramanian method with the Zhou asymmetric adapters, and to further optimize the configuration of the adapters. Ahn teaches certain advantages of using asymmetric adapters. The ordinary artisan would have been motivated to incorporate the Zhou adapters into the Balasubramanian method with the expectation that doing so would result in the advantages described in Ahn as to improved error correction, accuracy and sensitivity of the method. The ordinary artisan would have been further motivated to optimize the configuration of the adapters to customize the adapters as desired through routine experimentation. The ordinary artisan would have had an expectation of success as the design and modification of adapters is well-known in the art.
Regarding dependent claim 63, Balasubramanian additionally teaches that the DNA molecules comprise insert DNA from a subject, and that the method further comprises determining a likelihood that the subject has cancer (paras. 5, 88, 132, 218, 222; claims 132-133).
Regarding dependent claims 73 and 76, Balasubramanian additionally teaches identifying positions that were methylated in the DNA molecules and identifying positions that were hydroxymethylated in the DNA molecules, and identifying a genetic sequence of the DNA molecules (Figs. 1A-B, 2A-B; paras. 90, 96).
Regarding dependent claim 79, Balasubramanian additionally teaches that the synthesizing first complementary strands which are complementary to the first strands and second complementary strands which are complementary to the second strands: (a) comprises extending primers with dNTPs that are not methylated; (b) converts at least one methylated CpG to a hemimethylated CpG; and/or (c) converts at least one hydroxymethylated CpG to a hemihydroxymethylated CpG (Figs. 1A-B, 2A-B, 22, 48-49; para. 10).
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Balasubramanian (WO 2022/023753 A1) in view of Zhou (US Patent 2021/0404007 A1) and Ahn (Asymmetrical barcode adapter-assisted recovery of duplicate reads and error correction strategy to detect rare mutations in circulating tumor DNA, Scientific Reports, 7:46678, 1-9, 2017).
Regarding independent claim 2, Balasubramanian teaches …
A method of analyzing DNA molecules in a sample, the DNA molecules comprising first and second strands and adapters, the method comprising: a) synthesizing first complementary strands which are complementary to the first strands and second complementary strands which are complementary to the second strands (Figs. 1A-B, 2A-B);
b) glucosylating a 5-hydroxymethylated cytosine in at least one first or second strand before or after synthesizing the first and second complementary strands (Figs. 1A-B, 2A-B);
c) methylating a cytosine in at least one first complementary strand or second complementary strand, wherein the methylation converts a hemimethylated CpG to a fully methylated CpG (Figs. 1A-B, 2A-B; para. 87);
d) deaminating an unmodified cytosine, optionally with bisulfite conversion, in at least one first or second strand, thereby producing treated DNA molecules (Figs. 1A-B, 2A-B; para. 4);
and e) sequencing at least a portion of the treated DNA molecules (Figs. 1A-B, 2A-B).
Balasubramanian does not teach that the adapters are asymmetric. However, Zhou teaches asymmetric adapters, optionally Y-shaped adapters (paras. 19, 56; Figs. 2, 6B-C). Zhou additionally teaches that each asymmetric adapter comprises a deamination-sensitive cytosine that is an unmethylated cytosine (para. 20).
Further, Ahn teaches that the use of asymmetrical adapters provides advantages in NGS sequencing, including error correction, improved accuracy and sensitivity in variant calling and reduced false positives (e.g., p. 2, Results section; p. 6, para. 3; p. 7, paras. 3-4).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Balasubramanian method with the Zhou asymmetric adapters, and to further optimize the configuration of the adapters. Ahn teaches certain advantages of using asymmetric adapters. The ordinary artisan would have been motivated to incorporate the Zhou adapters into the Balasubramanian method with the expectation that doing so would result in the advantages described in Ahn as to improved error correction, accuracy and sensitivity of the method. The ordinary artisan would have been further motivated to optimize the configuration of the adapters to customize the adapters as desired through routine experimentation. The ordinary artisan would have had an expectation of success as the design and modification of adapters is well-known in the art.
Claims 7, 13, 15-16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Balasubramanian (WO 2022/023753 A1) in view of Zhou (US Patent 2021/0404007 A1) and Ahn (Asymmetrical barcode adapter-assisted recovery of duplicate reads and error correction strategy to detect rare mutations in circulating tumor DNA, Scientific Reports, 7:46678, 1-9, 2017).
Regarding independent claim 7, Balasubramanian teaches …
A method of analyzing DNA molecules in a sample, the DNA molecules comprising first and second strands and adapters, the method comprising: a) oxidizing a 5-hydroxymethylated cytosine in at least one first or second strand to 5-formylcytosine (Fig. 2D; paras. 96, 176, 178-180);
b) synthesizing first complementary strands which are complementary to the first strands and second complementary strands which are complementary to the second strands (Figs. 1A-B, 2A-B);
c) methylating a cytosine in at least one first complementary strand or second complementary strand, wherein the methylation converts a hemimethylated CpG to a fully methylated CpG (Figs. 1A-B, 2A-B; para. 87);
d) converting a modified cytosine in at least one first or second strand to a base read as a thymine, thereby producing treated DNA molecules (para. 96; Fig. 3);
and e) sequencing at least a portion of the treated DNA molecules (Figs. 1A-B, 2A-B).
Balasubramanian does not teach that the adapters are asymmetric. However, Zhou teaches asymmetric adapters, optionally Y-shaped adapters (paras. 19, 56; Figs. 2, 6B-C).
Further, Ahn teaches that the use of asymmetrical adapters provides advantages in NGS sequencing, including error correction, improved accuracy and sensitivity in variant calling and reduced false positives (e.g., p. 2, Results section; p. 6, para. 3; p. 7, paras. 3-4).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Balasubramanian method with the Zhou asymmetric adapters, and to further optimize the configuration of the adapters. Ahn teaches certain advantages of using asymmetric adapters. The ordinary artisan would have been motivated to incorporate the Zhou adapters into the Balasubramanian method with the expectation that doing so would result in the advantages described in Ahn as to improved error correction, accuracy and sensitivity of the method. The ordinary artisan would have been further motivated to optimize the configuration of the adapters to customize the adapters as desired through routine experimentation. The ordinary artisan would have had an expectation of success as the design and modification of adapters is well-known in the art.
Regarding dependent claims 13, 15-16 and 18, Balasubramanian additionally teaches that the method comprises converting a methylcytosine to carboxylcytosine, optionally with a TET enzyme, optionally TET1, TET2 or TET3, as part of converting the modified cytosine in at least one first or second strand to a base read as thymine (paras. 96, 149; Fig. 3). Balasubramanian additionally teaches that the method comprises reducing the carboxylcytosine, optionally by contacting with a reducing agent, optionally borane or a borohydride reducing agent, as part of converting the modified cytosine in at least one first or second strand to a base read as thymine, optionally wherein the carboxylcytosine is reduced to dihydrouracil (paras. 96-97, Fig. 3).
Claims 8, 10 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Balasubramanian (WO 2022/023753 A1) in view of Zhou (US Patent 2021/0404007 A1) and Ahn (Asymmetrical barcode adapter-assisted recovery of duplicate reads and error correction strategy to detect rare mutations in circulating tumor DNA, Scientific Reports, 7:46678, 1-9, 2017).
Regarding independent claim 8 and dependent claims 10 and 12, Balasubramanian
teaches …
A method of analyzing DNA molecules in a sample, the DNA molecules comprising first and second strands and adapters, the method comprising: a) oxidizing a 5-hydroxymethylated cytosine in at least one first or second strand to 5-formylcytosine, optionally by contacting with a ruthenate, optionally KRuO4 (Fig. 2D; paras. 96, 176, 178-180);
b) synthesizing first complementary strands which are complementary to the first strands and second complementary strands which are complementary to the second strands (Figs. 1A-B, 2A-B);
c) methylating a cytosine in at least one first complementary strand or second complementary strand, wherein the methylation converts a hemimethylated CpG to a fully methylated CpG (Figs. 1A-B, 2A-B; para. 87);
d) converting a modified cytosine in at least one first or second strand to a base read as a thymine, optionally by oxidizing a hydroxymethyl cytosine (para. 96; Fig. 3);
and e) sequencing at least a portion of the treated DNA molecules (Figs. 1A-B, 2A-B).
Balasubramanian does not teach that the adapters are asymmetric. However, Zhou teaches asymmetric adapters, optionally Y-shaped adapters (paras. 19, 56; Figs. 2, 6B-C). Zhou additionally teaches that each asymmetric adapter comprises an unmodified cytosine (para. 20).
Further, Ahn teaches that the use of asymmetrical adapters provides advantages in NGS sequencing, including error correction, improved accuracy and sensitivity in variant calling and reduced false positives (e.g., p. 2, Results section; p. 6, para. 3; p. 7, paras. 3-4).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Balasubramanian method with the Zhou asymmetric adapters, and to further optimize the configuration of the adapters. Ahn teaches certain advantages of using asymmetric adapters. The ordinary artisan would have been motivated to incorporate the Zhou adapters into the Balasubramanian method with the expectation that doing so would result in the advantages described in Ahn as to improved error correction, accuracy and sensitivity of the method. The ordinary artisan would have been further motivated to optimize the configuration of the adapters to customize the adapters as desired through routine experimentation. The ordinary artisan would have had an expectation of success as the design and modification of adapters is well-known in the art.
Conclusion
Claims 1-3, 6-8, 10, 12-13, 15-16, 18, 20-23, 25, 63, 69-70, 73, 76, 79 and 85 are being examined and are rejected. Claims 7-8 are objected to. No claims are allowed.
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/CAROLYN L GREENE/Primary Examiner, Art Unit 1681
1 Balasubramanian was cited in the Information Disclosure Statement submitted December 3, 2025.
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