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 .
Applicant’s amendment filed on December 19, 2025 is acknowledged and has been entered. Claim 1 has been amended. Claim 10 has been canceled. Claims 1-9 and 11-14 are pending.
Claims 1-9 and 11-14 are discussed in this Office action.
All of the amendments and arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons discussed below. Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
This action is made FINAL as necessitated by Amendment.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on October 22, 2025 was filed in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Priority
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 61448547, 61462972, 61634431, 61675020, 61571248, 61683331, 61398159, 61395850, fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. While each of these priority documents include support for universal adaptors or universal amplification and plural loci, none of these priority documents include sufficient (or any) support for the inclusion of epigenetic features, methylation analysis as associated with epigenetic analysis, or the inclusion of cancer or tumor or that epigenetic features are associated with tumors or cancer. Further, the priority document of 61426208 provides no support for cancer or tumors, or application of the method as claimed to analysis of cancer or tumor samples. Finally, the priority documents 61516996, 61982245, 61987407, 62066514, 62146188, 62147377 provide partial support for the claimed method, including cancer and methylation but make no mention at all of epigenetic features, as claimed. Further, the US Patent 8825412 does not provide support for the number of 1024 molecular barcodes, as amended. Therefore, as the priority documents of US Patent 10017812 and 61542508, for example, provide full support for the method as claimed, the claims are afforded an earliest priority of October 3, 2011.
New Grounds of Rejection as Necessitated by Amendment
Claim Interpretation
The amendment to the claim now recites “molecular barcode” and an additional step that recites “wherein the extracted cell-free DNA from the biological sample is tagged with a plurality of different molecular barcodes”. The specification does not specifically define molecular barcodes in a way that is structurally distinct from a “sample barcode”. The specification, instead, states the molecular barcode “contains the randomly generated molecular barcode” (paragraph 128). Further, the specification describes “molecular barcoding primers may consist of a sequence that is not complementary to the target sequence followed by random molecular barcode region followed by a target specific sequence” (paragraph 502).
The claim language of molecular barcode, in the context of claim 1, will be interpreted as reading on a tag or barcode or label that includes a random sequence or other “unique” sequence that is either distinguishable, distinct or unique and is not used to label entire samples for pooling.
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.
Claim(s) 1-3, 6-9 and 11-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Van Eijk et al. (US PgPub 20130137587; May 2013), Kivioja et al. (Nature Proceedings, April 2011, p 1-18, IDS reference, cited previously as relevant), Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189) and Gormally et al. (Mutation Research, 2007, 635:105-117).
With regard to claim 1, Van Eijk teaches a method for preparing a DNA fraction from a biological sample of a subject useful for analyzing genetic or epigenetic features associated with cancer, comprising:
(b) preparing a DNA fraction by: introducing at least 1024 adaptors each containing a universal priming sequence and a different molecular barcode into at least a subset of the extracted cell-free DNA molecules to produce a population of adapted DNA molecules,
wherein the at least a subset of the extracted cell-free DNA molecules from the biological sample are tagged with at least 1024 different molecular barcodes (paragraph 39 "large number of unique combinations is created by using at least two different nucleotide sequence identifiers that are incorporated in each sample"; Table 1 for a number of mathematically optimal situations for two NSis, which includes unique combinations of up to 16384);
performing universal amplification on at least some of the population of adapted DNA molecules using the universal priming sequence to produce an amplified adapted DNA molecules (paragraph 17-18, where paragraph 17 is focused on amplification with primers UT1/UT2 - universal tails, BC1/BC2barcode part 1, part 2 as depicted in Fig 11; see also where paragraph 18, where ligation of a pair of barcoded adapters is described as depicted in Fig 12 and using the same format as in paragraph 17), and
(c) performing massively parallel sequencing on the enriched DNA or its derivative to obtain sequence reads from at least a portion of the one or more target loci of interest (paragraph 31 and 34, where sequencing is described as including massively parallel sequencing)
With regard to claim 6, Van Eijk teaches a method of claim 1, wherein the selectively enriching comprises targeted multiplex amplification (paragraph 74, where multiplex PCR prioducts can be included).
With regard to claim 8, Van Eijk teaches a method of claim 7, wherein the selectively enriching further comprises amplifying the captured DNA using a second universal amplification (paragraph 17-18, where paragraph 17 is focused on amplification with primers UT1/UT2 - universal tails, BC1/BC2barcode part 1, part 2 as depicted in Fig 11; see also where paragraph 18, where ligation of a pair of barcoded adapters is described as depicted in Fig 12 and using the same format as in paragraph 17).
With regard to claim 9, Van Eijk teaches a method of claim 8, wherein the second universal amplification introduces a sample-specific barcode (claim 9, as published).
With regard to claim 11, Van Eijk teaches a method of claim 1, wherein the universal amplification introduces a sample-specific barcode, and wherein the enriched DNA of multiple samples are pooled together and sequenced in the same sequencing run (paragraph 4, 58, where samples are pooled and “During the sample preparation and/or during the high throughput sequencing method used, the different samples or part of the different samples may be pooled such that steps that may be performed simultaneously can be performed simultaneously. The sample origin may still be determined as no samples or part of different samples are pooled by which the sample origin may no longer be traced back”).
With regard to claim 13, Van Eijk teaches a method of claim 1, wherein at least one of the one or more target loci of interest is selected from the group consisting of: a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), an indel, a methylation site, and a copy number variation (CNV) (paragraph 74, where the method of sequencing can be used for “amplicon sequencing ( e.g. detection of mutations, natural polymorphisms), multiplexed SNP genotyping involving PCR primers such as KASP primers, Scorpions primers etc”).
Regarding claim 1, while van Eijk teaches tagging with barcodes, van Eijk does not teach (d) identifying from at least a portion of the sequence reads those derived from a same extracted cell-free DNA molecule in the at least a subset of the extracted cell-free DNA molecules using the tagged molecular barcode and (e) identifying from at least a portion of the sequence reads specific nucleic acid features.
With regard to claim 1, Kivioja teaches (d) identifying from at least a portion of the sequence reads those derived from a same extracted cell-free DNA molecule in the at least a subset of the extracted cell-free DNA molecules using the tagged molecular barcode (Abstract; p 2-3, Fig 1a which describes the inclusion of molecular barcodes during library generation; p 6 where the method of labeling with molecular barcodes is described; where it is noted that the molecular barcodes are described as umis) and
(e) identifying from at least a portion of the sequence reads specific nucleic acid features (Abstract; p 2-3, Fig 1a which describes the inclusion of molecular barcodes during library generation; p 6 where the method of labeling with molecular barcodes is described; where it is noted that the molecular barcodes are described as umis).
Regarding claim 1, while Van Eijk teaches the analysis, amplification and sequencing of nucleic acids, Van Eijk does not teach the step wherein cell-free DNA is extracted from the biological sample. Also, regarding claims 1 and 7, while Gnirke teaches massively parallel sequencing and while Van Eijk mentions methylation, Van Eijk is not specific in detecting epigenetic features associated with cancer using the method, as claimed.
With regard to claim 1, Gormally teaches
(a) extracting cell-free DNA molecules from the biological sample (p 107, section 2 “2. Circulating free plasma DNA: characteristics and origins and 2.1. Occurrence of CFDNA, where cell-free DNA is isolated from blood; see also p 107, section “2.2. Tumoral origin of CFDNA in cancer patients” where cell free DNA is analyzed for tumors as well) and (d) from the sequence reads of one or more genetic or epigenetic features associated with cancer wherein sequence reads derived from the same extracted cell-free DNA molecule are identified using the tagged molecular barcode (Abstract, p 110, col. 2 section 3.3 Detection of genetic and epigenetic changes in CFDNA, Table 1, which describes a selection of genetic and epigenetic changes in tumors and cell free DNA, including hypermethylation of CDKN2a as described on p 112-113).
With regard to claim 2, Gormally teaches a method of claim 1, wherein the biological sample is a blood, plasma, serum, or urine sample (p 107, section 2 “2. Circulating free plasma DNA: characteristics and origins and 2.1. Occurrence of CFDNA, where cell-free DNA is isolated from blood).
With regard to claim 3, Gormally teaches a method of claim 1, wherein the genetic or epigenetic features associated with cancer comprises single nucleotide polymorphism or variant, copy number variation, insertion, deletion, or nucleotide methylation (Abstract, p 110, col. 2 section 3.3 Detection of genetic and epigenetic changes in CFDNA, Table 1, which describes a selection of genetic and epigenetic changes in tumors and cell free DNA, including hypermethylation of CDKN2a as described on p 112-113).
With regard to claim 12, Gormally teaches a method of claim 1, wherein the cell-free DNA molecules comprises cancer DNA molecules, and wherein the method further sequence comprises estimating the fraction of cancer DNA molecules in the cell-free DNA based on the reads (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632; see also paragraph 493, where analysis of wild type to mutation is described).
With regard to claim 14, Gormally teaches a method of claim 1, wherein the one or more target loci of interest includes two target loci of interest, one of which contains a single nucleotide variation (SNV) and another of which contains a nucleotide methylation site (Abstract, p 110, col. 2 section 3.3 Detection of genetic and epigenetic changes in CFDNA, Table 1, which describes a selection of genetic and epigenetic changes in tumors and cell free DNA, including hypermethylation of CDKN2a as described on p 112-113).
Regarding claim 1 and 7, while Van Eijk teaches the method of universal amplification and massively parallel sequencing, van Eijk does not specifically teach enrichment of short nucleic acids, van Eijk also does not teach the use of hybrid capture probes.
With regard to claim 1, Gnirke teaches selectively enriching for at least a subset of the amplified adapted DNA molecules or its derivative containing one or more target loci of interest to produce enriched DNA (Abstract, Figure 1, where hybrid capture probe enrichment is described).
With regard to claim 7, Gnirke teaches a method of claim 1, wherein the selectively enriching comprises capturing at least some of the amplified adapted DNA molecules comprising one or more loci using hybrid capture probes (Abstract, Figure 1, where hybrid capture probe enrichment is described).
Further, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of van Eijk to include the hybrid capture probes, enrichment and massively parallel sequencing techniques as taught by Gnirke to arrive at the claimed invention with a reasonable expectation for success. Van Eijk teaches a method of high throughput sequencing. While van Eijk does not teach an enrichment step, van Eijk is focused on a method which “may simplify sample preparation, may reduce the workload, may optimize technical performance and can reduce costs” (paragraph 6). Gnirke teaches specific library-based enrichment of nucleic acids with the inclusion of hybrid capture probes. Gnirke teaches “We developed a capture method that uses biotinylated RNA ‘baits’ to fish targets out of a ‘pond’ of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target 415,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out”. Gnirke also teaches “Uniformity of capture, along with specificity, is the main determinant for the efficiency and practical utility of any bulk enrichment
method for targeted sequencing” (p 185 “evenness of coverage” heading). Gnirke concludes “have developed a hybrid-selection method for enriching specific subsets of a genome that is flexible, scalable and efficient” (p. 186, Discussion heading). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of van Eijk to include the hybrid capture probes and enrichment as taught by Gnirke to arrive at the claimed invention with a reasonable expectation for success.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of Van Eijk to include the molecular barcode teaching of Kivioja to arrive at the claimed invention with a reasonable expectation for success. While Van Eijk teaches a method that includes barcode labeling, Van Eijk does not look to tracing individual reads as described in steps d) and e) of the method. Kivioja teaches “In this method, each individual molecule of interest is first made unique (Fig. 1a). This can be accomplished for example by taking a small aliquot, by fragmentation or by addition of a random DNA sequence label. Any combination of these manipulations can be used to generate a library of molecules where each molecule has a distinct sequence” (p 2). Kivioja also teaches “As long as the complexity of the library is maintained, it can be (differentially) amplified, normalized and otherwise processed without loss of information about how many molecules were originally present in the sample. This is because making each molecule different from each other during the library generation step stores the information about the original number of DNA molecules into a molecular memory consisting of the number of distinct sequences (umis) in the library (Fig. 1a)” (p 2-3). Finally, Kivioja teaches “Whereas measuring the number of copies of each sequence is difficult, counting the number of distinct sequences (umis) is trivial, and this information is not lost during amplification or any other complexity-preserving manipulation of the library. Sequencing of the library is then used to determine the absolute number of DNA molecules of each species in the original sample (Fig. 1a). When enough sequences have been obtained, each umi will have been observed multiple times, and the number of original DNA molecules can be determined simply by counting the number of umis” (p 3). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Van Eijk to include the molecular barcode teaching of Kivioja to arrive at the claimed invention with a reasonable expectation for success.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of van Eijk and Gnirke to apply the method to detection of cancer and for detection of epigenetic features associated with cancer focused on cell free nucleic acids as described by Gormally to arrive at the claimed invention with a reasonable expectation for success. First, van Eijk specifically teaches the method of sequencing is useful for “amplicon sequencing ( e.g. detection of mutations, natural polymorphisms), multiplexed SNP genotyping involving PCR primers such as KASP primers, Scorpions primers etc” (paragraph 74). Next, Gormally teaches “the technical issues involved in obtaining, using and analyzing CFDNA in cancer or healthy subjects. We also summarize the literature available on the mechanisms of CDNA release as well as on cross-sectional or prospective studies aimed at assessing the clinical and biological significance of CFDNA. These studies show that, in some circumstances, CFDNA alterations are detectable ahead of cancer diagnosis, raising the possibility of exploiting them as biomarkers for monitoring cancer occurrence” (Abstract). Finally, Gormally also teaches “Presence in CFDNA of genetic or epigenetic alterations, such as mutations in KRAS2, MI at various loci and hypermethylation of CDKN2A, have been shown to indicate disease recurrence in patients who have undergone cancer curative therapies” (p. 112, col. 2 in “4.1. Associations between altered CFDNA and disease in cross-sectional studies”). Gormally also teaches “the analysis of CFDNA for the follow-up of patients who have undergone curative therapy appears promising. The possibility of identifying genetic and epigenetic alterations in a biopsy or in the resected tumor specific to the patients and to regularly check for the presence of these alterations in CFDNA might allow early detection of disease Recurrence” (p. 113, col. 1 in “4.1. Associations between altered CFDNA and disease in cross-sectional studies”). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of van Eijk and Gnirke to apply the method to detection of cancer and for detection of epigenetic features associated with cancer as described by Gormally to arrive at the claimed invention with a reasonable expectation for success.
Claim(s) 4-5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Van Eijk et al. (US PgPub 20130137587; May 2013), Kivioja et al. (Nature Proceedings, April 2011, p 1-18, IDS reference, cited previously as relevant), Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189) and Gormally et al. (Mutation Research, 2007, 635:105-117) as applied over claims 1-3, 6-9 and 11-14 above and further in view of Pieprzyk et al. (US PgPub 20140186827; July 2014).
With regard to claim 4, Pieprzyk teaches a method of claim 1, wherein step (b) comprises selectively enriching for 1,000- 500,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
With regard to claim 5, Pieprzyk teaches a method of claim 1, wherein step (b) comprises selectively enriching for 10,000- 200,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
An ordinary practitioner would have recognized that the results optimizable variables of time, product amount and number of loci enriched and which can be analyzed could be adjusted to maximize the desired results. As noted in In re Aller, 105 USPQ 233 at 235,
More particularly, 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.
Routine optimization is not considered inventive and no evidence has been presented that the number of sites detected and sequenced was other than routine, that the products resulting from the optimization have any unexpected properties, or that the results should be considered unexpected in any way as compared to the closest prior art.
Further, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of Van Eijk, Gnirke and Gormally to include high level multiplex as described by Pieprzyk to arrive at the claimed invention with a reasonable expectation for success. Pieprzyk teaches “It will be recognized that, in certain embodiments, a large number of different target sequences ( e.g., 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, 50 or more, 100 or more per chromosome or other template(s )), can be tagged. Moreover using various tagging strategies, different amplification produces are readily discriminated thereby permitting the methods to be highly multiplexed” (paragraph 218). Pieprzyk also teaches “In certain embodiments, multiplex detection is carried out in individual amplification mixture, e.g., in individual reaction chambers of a microfluidic device, which can be used to further increase the number of samples and/or targets that can be analyzed in a single assay or to carry out comparative methods, such as comparative genomic hybridization (CGH). In various embodiments, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10000 or more amplification reactions are carried out in each individual reaction chamber” (paragraph 295). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Van Eijk, Gnirke and Gormally to include high level multiplex as described by Pieprzyk to arrive at the claimed invention with a reasonable expectation for success.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1-9 and 11-14 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Citation of Pertinent Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Anker et al. (Cancer and Metastasis Reviews, 1999, 18:65-73). Sasaki et al. (Biochem Biophys Res Comm, 2003, 309:305-309). Miner et al. (Nucleic Acids Research, 2004, 32(17): 1-4, IDS reference).
Conclusion
No claims are allowed. All claims stand rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHANIE KANE MUMMERT whose telephone number is (571)272-8503. The examiner can normally be reached M-F 9:00-5:30.
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/STEPHANIE K MUMMERT/Primary Examiner, Art Unit 1681