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 .
Information Disclosure Statement
The IDS received on February 12, 2025 and July 9, 2026 are proper and are being considered by the Examiner.
Drawings
The drawings received on June 20, 2024 are acceptable.
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.
Claim 80 is 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 80 is indefinite because the claim simply recites that which is implicitly required. Claim 80 depends from claim 1. Claim 1 necessarily requires that the polymerase incorporate a cytosine analog for the method to work. Claim 80 therefore, simply recites that is implicit of the parent claim and therefore does not add anything more to the parent claim.
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.
Claims 1, 2, 5, 6, 22, 25, 28, 30, 33, 37, 38, 65, 74, 75, 80, and 145-147 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kester, H.A. (US 2015/0011408 A1, published January 8, 2015).
With regard to claim 1, Kester teaches a method of detecting genetic and epigenetic information in a single-workflow (“the resulting double-stranded product represents the exact genomic sequence, where cytosines are methylated, while the other part is information in the determination of methylation status”, section [0078]), wherein an embodiment of the method is represented by the below-reproduced Figure (from Fig 1B):
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As shown, Kester teaches the steps of: a) providing a plurality of first single-stranded DNA fragments (“providing a sample comprising a template nucleic acid”, section [0012], also “[i]n certain aspects, the template nucleic acid is DNA. In certain aspects, the template nucleic acid is single-stranded”, section [0027]); b) subjecting the plurality of first single-stranded DNA fragments to a round of primer extension in the presence of: i) a primer that anneal to at least a portion of the first single-stranded DNA fragments (see above primer, also “a primer is annealed to the template strand via a region of complementarity”, section [0078]); ii) a nucleic acid polymerase (“[a] nucleic acid polymerase is then used to extend the oligonucleotide primer”, section [0078]); and iii) a mixture of nucleotides comprising a cytosine analog that is resistant to cytosine conversion; thereby generating a plurality of first strands corresponding to the first single-stranded DNA fragments and a plurality of second strands that are complementary to the first strands, wherein the second strands comprise the cytosine analog that is resistant to cytosine conversion (“extend the oligonucleotide primer in the presence of a bisulfite-resistant cytosine analog such as 5-methyl dCTP”, section [0078]); c) subjecting the pluralities of first and second strands to cytosine conversion treatment under conditions such that unmethylated cytosine(s), if present in the first strands undergo cytosine conversion (“conversion treatment to convert cytosine residues in the template nucleic acid into residues comprising the other base, resulting in a converted template nucleic acid and a non-converted nucleic acid template; “, section [0012], also “obtaining bisulfite-converted template nucleic acid which may or may not comprise at least one uracil residue”, section [0082]); and d) detecting methylation information from at least a portion of the plurality of the first stands and detecting sequence information from at least a portion of the plurality of the second strands (“determining the nucleotide sequence of the bisulfite-converted template nucleic acid; determining the nucleotide sequence of the bisulfite-converted complementary copy; and comparing the nucleotide sequence of the bisulfite-converted complementary to the nucleotide sequence of the bisulfite-converted template nucleic acid, thereby determining the nucleotide sequence of the template nucleic acid prior to bisulfite-conversion and the methylation status of the template nucleic acid prior to bisulfite conversion”, section [0039]).
With regard to claim 2, the detecting is by sequencing (“method of sequencing nucleic acid comprising cytosine is provided”, section [0012], also “oligonucleotides on the 5’ and 3’ ends are used for priming sequencing reactions …”, section [0049], “methods of sequencing nucleic acids and determining the methylation level and/or patterns of the nucleic acids”, section [0054]).
With regard to claims 5 and 6, the targets are amplified via PCR (“amplification of the targes using PCR … may also be done … particularly useful when the target sequence is present in the sample at extremely low copy numbers”, section [0102]1).
With regard to claim 22, Kester teaches the ligation of an adapter to a first strand nucleic acid which is complementary to a sequencing primer that is to be used:
“adapter oligonucleotides may be ligated to the template and complementary nucleic acid molecules. The adapter oligonucleotides, can then be used to identify the bisulfite-converted template and the bisulfite-converted complementary copy as members of a nucleic acid pair” (section [0118])
“For example, a sequencing primer can be annealed to a priming site that was ligated to a target sequence prior to bisulfite treatment” (section [0132])
Based on this teaching, with regard to claims 25, 28, 30, 33, and 37, the first adapter sequence on the first strand and its second, complementary strand that has been polymerized with methylated cytosines will result in the adapter of the first strand being converted when treated with bisulfite, while the second complementary strand that has been polymerized with 5’ methyl-cytosine (thus not converted), becoming different in their sequence and therefore becoming not fully complementary.
With regard to claim 38, the plurality of first and/or second strands are amplified (“[b]isulfite-treated nucleic acids, such as DNA, can subsequently be analyzed by conventional molecular techniques, such as PCR amplification, sequencing, and detection”, section [0120]).
With regard to claims 65 and 145, the sample is obtained from cells (“DNA sample used … obtained from any biological fluid, cell, tissue, organ or portion thereof, that contains genomic DNA suitable for methylation detection … neoplastic cell … colon … breast … ovary … prostate …”, section [0161]).
With regard to claim 74, the cytosine analog is 5-methyl dCTP (“bisulfite-resistant cytosine analog can be selected from the group consisting of: 5’ethyl dCTP; 5-methyl dCPT … 5-aza-dCTP …” , section [0016]).
With regard to claim 75, the cytosine conversion is bisulfite treatment (see above).
With regard to claim 80, the polymerase incorporates the modified cytosine (see above).
With regard to claim 146, the methylation level and sequence are analyzed for cancer (“in particular embodiments a disease or condition to be analyzed with respect to methylation levels is cancer …”, section [0168]).
With regard to claim 147, prognostics regarding therapy is also disclosed (“prognostic methods can be used to identify surgically treated patients likely to experience cancer recurrence so that they can be offered additional therapeutic opinions … such as chemotherapy, radiation … determining a proper course of treatment for a patient having cancer”, section [0171]).
Therefore, Kester anticipates the invention as claimed.
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 11, 13, 14, 21, 60, and 148-151 are rejected under 35 U.S.C. 103 as being unpatentable over Kester, H.A. (US 2015/0011408 A1, published January 8, 2015) in view of Horn, Susanne (Ancient DNA: Methods and protocols, 2011, vol. 840, pages 177-188).
The teachings of Kester have already been discussed above.
Kester, while teaching that the method employs amplification of the template nucleic acids, as well as amplifying the bisulfite-treated products (“[b]isulfite-treated nucleic acids, such as DNA, can subsequently be analyzed by conventional molecular tehcniques, such as PCR amplification, sequencing, and detection”, section [0120]), do not explicitly teach that the products should be enriched (claim 11), or that the first and second strand products are separated (claim 13), wherein the separation is effected via bait molecules (claim 14).
While Kester explicitly teaches subjecting the first strand to primer extensions, the artisan does not explicitly teach that two or more rounds of primer extension is performed (claim 21).
While Kester explicitly teaches sequencing, do not explicitly teach that the first strands is sequenced at a different depth than that of the second strands (claim 60).
While Kester explicitly teaches that the method of the disclosure reveals the sequence of the template nucleic acid and the methylation status, as well as applying the method for prognostics/diagnostics pertaining to cancer, the artisan does not explicitly teach that mutational status (somatic mutation) and MRD is determined (claims 148 and 149), or that the mutation is germline mutation (claim 150), or the sequence information pertains to LOH in HLA genes or mutational burden (claim 151).
Horn teaches a well-known means of enriching for target via a DNA capture:
“In many ancient DNA (aDNA) extracts, DNA fragments representing the target loci are present at very low copy-number compared to sequences of contaminating exogenous DNA. Such experiments therefore require an enrichment step, where the amount of target DNA is increased in a library to be sequenced, relative to nontarget DNA” (page 177)
Horn also teaches various means known in the art for the enrichment of target DNAs utilizing DNA bait molecules (see Table 2, page 180)
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kester and Horn, thereby arriving at the invention as claimed for the following reasons.
Preliminarily, with regard to applying the sequencing method of Kester that reveals the methylation status as well as the sequence of the target DNA, applying this method for the prognostics/diagnostics pertaining to cancer, such as determining mutational status (somatic mutation) and MRD, or germline mutation, or the sequence information pertains to LOH in HLA genes or mutational burden, such applications have been well-known and practiced in the art as methylation status and mutational status has been well-established to be correlated with cancer, in determining minimal residual disease (MRD), mutational burden in samples of tumors, as well as HLA gene matching. Therefore, one of ordinary skill in the art would have had a reasonable expectation of success that the sequence information produced from Kester’s method would have been useful for the same purposes.
As regards to the combination of enrichment step utilizing bait molecules for isolating the target DNA molecules, one of ordinary skill in the art would have been motivated to do so for the purpose of separating the first strand DNA templates whose unmethylated cytosines have been converted during the bisulfite-treatment and the second strand DNA whose cytosines have not been converted so as to determine the sequences from the respective strands, allowing the generated sequences to be compared in determining the sequence of the target DNA as well as the methylation levels therein. Combining prior art known means of enrichment, such as the use of DNA baits would have yielded a predictable outcome for such a purpose, rendering the invention as claimed prima facie obvious.
As to subjecting the first single-stranded DNA fragments to two or more rounds of primer extension for producing a second, complementary strand comprising the incorporated methyl-cytosines, doing so would have been obvious, so as to provide additional strands to sequence and/or providing sufficient methyl-cytosine incorporation into the second strands when synthesized, the process of which would have been well-within the purview of ordinarily skilled artisan in view of optimizing the sequencing results.
Similarly, adjusting the sequencing depth of the first and second strands when sequencing would have been obvious in view of optimizing the sequence read results, as well as to compensate the well-known process of bisulfite treatment resulting in degradation of the treated DNA as well as reducing sequence complexity (from 4 bases down to 3 bases).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to Young J. Kim whose telephone number is (571) 272-0785. The Examiner can best be reached from 7:30 a.m. to 4:00 p.m (M-F). The Examiner can also be reached via e-mail to Young.Kim@uspto.gov. However, the office cannot guarantee security through the e-mail system nor should official papers be transmitted through this route.
If attempts to reach the Examiner by telephone are unsuccessful, the Examiner's supervisor, Gary Benzion, can be reached at (571) 272-0782.
Papers related to this application may be submitted to Art Unit 1681 by facsimile transmission. The faxing of such papers must conform with the notice published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 CFR 1.6(d)). NOTE: If applicant does submit a paper by FAX, the original copy should be retained by applicant or applicant’s representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED, so as to avoid the processing of duplicate papers in the Office. All official documents must be sent to the Official Tech Center Fax number: (571) 273-8300. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-1600.
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/YOUNG J KIM/Primary Examiner
Art Unit 1637 July 28, 2026
/YJK/
1 Beginning template nucleic acids do not have the second strand synthesized yet and therefore, the primer will only anneal to the first template strand in a PCR reaction.
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