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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 11/21/2025,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is FINAL
Claim Status
Applicant previously elected Group 1: claims 1-12 (1/27/2025) in response to Examiner Restriction (10/25/2024) with traverse. Applicant newly elected SEQ ID: NO 1 as recited in dependent claim 12 (11/21/2025).
Applicant cancelled claims 2 and 13-20 (6/20/2025). Claims 1, 3, 6, 8, 9, 11, and 12 have been amended (6/20/2025). No new matter was added. Claims 1 and 3-12 are under examination (6/20/2025).
Election/Restrictions
Applicant's election with traverse of SEQ ID NO: 1 in the reply filed on November 21, 2025 is acknowledged.
The restriction requirement (9/25/2025) is maintained because claim 12 recites at least one polynucleotide having a sequence selected from SEQ ID NOs: 1-50. Each SEQ ID corresponds to a distinct nucleotide sequence, and therefore constitutes a separate species within the claimed genus. The sequences are not linked by a single special technical feature that defines a common inventive concept over the prior art. As previously indicated, the shared concept of DNA methylation array comprising cross-species conserved CpG sequences is disclosed in the art (i.e., Feinberg et al. ), and thus does not qualify as a special technical feature under PCT Rules 13.1 and 13.2. Because the SEQ ID NOs represent patentably distinct embodiments requiring separate examination and potentially different prior art searches, unity of invention is lacking. See MPEP 821.03. The mere assertion that the species share a common inventive concept is insufficient where the alleged linking feature is known in the art and does not render the species distinct.
Claims 1 and 3-12 are under examination, and SEQ ID NO: 1 is elected for examination for dependent claim 12.
Priority
Claims 1 and 3-12 are given a priority date of 1/18/2019.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Information Disclosure Statements from 2/28/2025, 3/20/2025 and 2/2/2026 are considered.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames, as well as the embedded hyperlinks, are withdrawn in view of Applicant’s amendments.
Claims:
The minor formality objections to claims 3, 8, and 10-12 are withdrawn in view of Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1-7, 9 and 10-11 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s cancellation of claim 2 and amendments of claims 3, 8, and 10-12 to address indefiniteness and lack of antecedent basis.
Rejections Maintained
Claim Rejections - 35 USC § 101
Claims 1, 3-8 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural phenomenon without significantly more.
The claims recite methods for making a DNA methylation array by performing polynucleotide sequence alignment comparing a human genome with non-human mammalian genomes, ranking the polynucleotide sequences based on homology criteria, selecting polynucleotides that cross-hybridize to sequences in non-human mammalian species, and coupling selected sequences to a matrix to form an array.
The claims are directed to a natural phenomenon because they are based on the naturally occurring relationship between homologous DNA sequences across different mammalian species and the natural conservation of CpG methylation sites. The correlation between sequence homology and methylation site conservation is a naturally occurring principle.
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
performing a polynucleotide sequence alignment comparing a human genome with non-human mammalian genomes to identify polynucleotide sequences with homologous CpG methylation sites;
ranking the polynucleotide sequences based on sequence homology criteria to sequences in non-human mammalian species;
selecting polynucleotides that cross-hybridize to sequences in non-human mammalian species with defined mismatch parameters;
coupling the selected sequences to a matrix to form a DNA methylation array.
Thus, the claims are directed to statutory categories (i.e., processes and machine).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
The claims recite a natural phenomenon. The natural phenomenon is the naturally occurring relationship between homologous DNA sequences across different mammalian species and the conservation of CG methylation sites. The correlation between sequence homology and methylation site conservation is a naturally occurring principle that exists independent of human action.
Further, the “mental process” of determining correlations between homologous DNA sequences across species and analyzing methylation conservation patterns corresponds to “an abstraction”, or an idea having no particular concrete or tangible form. Thus, the claimed invention describes a judicial exception, which corresponds to abstractions and natural principles.
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim limitations are considered to be; (a) a mental process of evaluating/interpreting data by identifying polynucleotide sequences with homologous CpG methylation sites across species, ranking based on sequence homology, and determining which sequences will cross-hybridize with specific mismatch parameters, and (b) natural principle of conservation of CpG methylation sites across evolutionary related mammalian species (i.e., natural principle).
While the claims recite steps of “performing polynucleotide sequence alignment,” “ranking the polynucleotide sequences,” “selecting a plurality of polynucleotides,” and “coupling selected sequences to a matrix”, these steps are recited at a high level of generality and amount to mere data gathering steps. There are no additional steps which apply the identified judicial exceptions into a practical application. Thus, the claims do not provide for any element/step that integrates the law of nature into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether ra claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” elements/steps of, “performing polynucleotide sequence alignment,” “ranking the polynucleotide sequences,” “selecting a plurality of polynucleotides,” and “coupling selected sequences to a matrix” are “physical” steps telling a practitioner to simply implement the natural phenomenon and abstract ideas and are considered to be within the purview of one in the art as being routine and conventional in the art when investigating conservation of DNA methylation sites across mammalian species.
For example, Ross discloses (WO 2012/034170 A1 published 3/22/2012) a method of making a DNA molecular or methylation array comprising a plurality of: nucleic acid molecules comprising a nucleotide sequence corresponding to individualized DNA markers or a functional derivative, fragment, variant or homologue for either human or non-human mammalian genomes (p. 19, lines 1-10). Ross also discloses a polynucleotide sequence alignment for scoring, specifically via blood DNA sample (i.e., human or non-human) that are separately ligated with bar coded "MID" linkers for later scoring or ranking via specified probes (p. 23, lines 25-30; p. 88, lines 15-20). Further, Ross discloses that these libraries or polynucleotide sequence alignment sets are segregated to individual samples using bar-code sequences and further aligned with bisulphite converted sequence of each amplicon, including the fraction of cytosines at each potential 5’-cytosine-phosphate-guanine-3’ (CpG) methylation site (p. 30, lines 1-10; p. 88, lines 20-25) via the methylation of the overall amplicon or individual CpG sites that are assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis within non-human mammalian genomes via individualized probes coupled to a matrix (p. 45, lines 20-25; p. 58, lines 5-15; p. 80, lines 5-10), establishing this array methodology as a conventional technique.
Further, Irizarry et al. (WO 2009/021141 Al, published 2/12/2009, from IDS 7/8/2021) discloses methods and devices for comprehensive methylome analysis utilizing high-throughput
arrays to determine relative methylation (Paragraph 1, lines 1-3). Irizarry specifically discloses a method of designing a microarray for DNA methylation analysis, including identifying CpG islands in a polynucleotide sequence and generating a plurality of probe sequences corresponding to the identified CpG islands (Paragraph 12, lines 1-5). Further, Irizarry discloses that a plurality of discrete genomic regions may be generated such that each genomic region includes more than one of the generated pluralities of probe sequences (Paragraph 12, lines 5-10), thus demonstrating a level of routine and convention for the use of microarrays to identify specific areas of CpG methylation sites for comparison of a variety of homologs.
Further, Gujar et al. (“Profiling DNA methylation differences between inbred mouse strains on the lllumina Human lnfinium Methylation EPIC microarray”, PLOS ONE; published 3/12/2018, from IDS 6/5/2023) discloses methods for using an Illumina™-based array to measure DNA methylation in mice via defined probes targeting conserved regions and for differential methylation analysis through comparisons between the array-based assay and affinity-based DNA sequencing of methyl-CpGs (MBD-seq) (Abstract). Further, Gujar discloses that CpG islands (CGis) are largely conserved between mice and humans and the two species share similar numbers of CGis in promoter regions of genes, therefore it is feasible that probes on the human microarrays that target these sites may have some application in research using rodent models (Introduction: Paragraphs 1-2), thus establishing a high level of routine and convention for the application of non-human mammalian homologs in DNA methylation microarrays.
Therefore, providing a DNA methylation array comprising a plurality of polynucleotides to identify CpG methylation sites that are homologs to specified sequences within genomes of non-human mammalian species was routine and conventional before the effective filing date of the claimed invention.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., “performing polynucleotide sequence alignment”, “ranking the polynucleotide sequences”, “selecting a plurality of polynucleotides”, and “coupling selected sequences to a matrix”) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to analyze conservation of CpG methylation sites across mammalian species. In context with the other recited claim limitations, the language “identifying homologous CpG methylation sites across species comprising: performing a polynucleotide sequence alignment comparing a human genome with a plurality of non-human mammalian genomes”; ranking based on sequence homology”; selecting polynucleotides that cross-hybridize”; and “coupling to a matrix” indicates whether or not the relationship/correlation between DNA sequence conservation and methylation site conservation exists.
This information simply tells a practitioner about the relevant natural law, at most adding a suggestion that the researcher should take those laws into account. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps are considered conventional and specified at a high level of generality. The claim limitations do not transform the natural phenomenon and abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the natural phenomenon with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
Applicant’s Response: The Applicant argues that the claims are not directed to a natural phenomenon, but instead to a practical application, specifically, a method of making a DNA methylation array comprising selected polynucleotides coupled to a physical matrix such as a bead or chip. The Applicant further contends that the claims integrate any alleged natural correlation into a concrete, man-made device formed from non-natural components arranged in a specific configuration. The Applicant also asserts that the methodology for selecting and assembling the array uses non-conventional techniques and therefore provides significantly more than a mere recitation of a natural principle.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
Although the claims are framed as a method of making a DNA methylation array, the focus of the claimed invention remains the natural correlation between sequence homology across mammalian species and CpG methylation site conservation. Under Step 2A, Prong Two, merely coupling selected polynucleotides to a bead or chip does not meaningfully integrate the judicial exception into a practical application, because the selection itself is driven entirely by the natural principle, and the matrix serves only as a generic implementation environment. The claimed “array” is thus the result of applying natural correlation using conventional laboratory components.
Further, the Applicant’s assertion that the methodology is non-conventional is unsupported in view of the record. Specifically, the cited prior art demonstrates that sequence alignment, ranking by homology, selecting probes based on mismatch criteria, and constructing methylation arrays were well-understood, routine, and conventional techniques at the time of filing. Under Step 2B, the ordered combination of these routine bioinformatics and array-construction steps does not supply an inventive concept, as it merely instructs practitioners to apply a natural conservation principle using standard tools.
Accordingly, the 101 rejection is maintained.
Claim Rejections - 35 USC § 102
Claims 1 and 7 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Ross et al. (WO 2012/034170 A1, published 3/22/2012).
Regarding claim 1, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that the DNA molecular or methylation array comprises a plurality of:
nucleic acid molecules comprising a nucleotide sequence corresponding to individualized DNA markers or a functional derivative, fragment, variant or homologue for either human or non-human mammalian genomes (p. 19, lines 1-10). Ross also teaches a polynucleotide sequence alignment for scoring, specifically via blood DNA sample (i.e., human or non-human) were separately ligated with bar coded "MID" linkers for later scoring or ranking (p. 23, lines 25-30; p. 88, lines 15-20). Ross teaches that these libraries or polynucleotide sequence alignment sets were segregated to individual samples using the bar-code sequences and aligned with the bisulphite converted sequence of each amplicon, including the fraction of cytosines at each potential 5’-cytosine-phosphate-guanine-3’ (CpG) methylation site was determined for each sample—including homologous proteins (p. 30, lines 1-10; p. 88, lines 20-25) via the methylation of the overall amplicon or individual CpG sites can then be assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis within non-human mammalian genomes (p. 45, lines 20-25). Ross also teaches that individual probes are composed of specified ranking criteria, representing the raw probe response value for the given combination of methylation status corresponding to methylation values in individual samples (p. 80, lines 5-10). Further, Ross teaches that gene-specific primers or individualized adapters are added to the ends of the randomly fragmented human DNA and the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, and further amplified using primers that cross-hybridize to the adaptor sequences within genomes of non-human mammalian species (p. 46, lines 10-15). Ross further teaches that the previously described individualized probes for ranking permit distinction between the ligated and unligated probes, in which case the presence of both labels in the same eluate fraction confirms the ligation event and the target nucleic acid is bound to a solid matrix (p. 58, lines 5-10).
Ross teaches that the previously described DNA methylation array is composed of polynucleotides from human genomes that even a single base pair mismatch compared to a non-human mammalian genome significantly alters the level of fluorescence detected in a sample (p. 60, lines 30-35).
Regarding claim 7, Ross teaches that the previously described DNA methylation array is comprised of nucleic acid bound to a solid matrix (i.e., Southern hybridization, slot blot, dot blot, or microchip assay format) allowing the presence of both the diagnostic and contiguous probes to be determined directly (p. 58, lines 5-15).
Ross teaches each and every limitation of claims 1-2 and 7, and therefore Ross anticipates claims 1-2 and 7.
Applicant’s Response: The Applicant argues that Ross does not anticipate the claims because it fails to teach the specific constellation of methods steps recited in amended claim 1. Specifically, the Applicant contends that Ross does not teach selecting polynucleotides by performing a sequence alignment comparing a human genome with at least five mammalian species, including at least one non-placental species, not selecting sequences with no more than a three-basepair mismatch across those species.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
Anticipation requires that each and every element of the claimed invention be disclosed, either expressly or inherently, in a single prior art reference (see MPEP 2131; MEHL/Biophile Int’l Corp v. Milgraum, 192 F. 3d 1362 (Fed. Cir. 1999)). Ross teaches a method of screening for modulation in DNA methylation using a DNA molecular or methylation array comprising a plurality of nucleic acid molecules corresponding to individualized DNA markers, including homologues from human and non-human mammalian genomes (p. 19, lines 1-10). Ross further teaches that performing polynucleotide sequence alignment for scoring and ranking (p. 23, lines 25-30; p. 88, lines 15-20), assessing CpG methylation at individual sites following bisulfite conversion (p. 30, lines 1-10; p. 88, lines 20-25), and utilizing probes that cross-hybridize with sequences in non-human mammalian genomes (p. 46, lines 10-15). Ross further teaches binding nucleic acids to a solid matrix in array formats (p. 58, lines 5-10), thereby satisfying the coupling to a matrix limitation.
The Applicant specifically argues that Ross does not teach (1) comparing a human genome with at least five non-human mammalian species including at least one non-placental species, and (2) selecting sequences having no more than a three basepair mismatch across those species. As highlighted above, Ross teaches cross-species homologous sequences and cross-hybridization to non-human mammalian genomes, and teaches ranking and probe discrimination based on hybridization characteristics. Where a reference discloses a genus (non-human mammalian genomes) that encompasses the claimed species grouping, anticipation is not avoided merely because the reference does not enumerate a specific minimum number of species. See MPEP 2131.02. Moreover, Ross teaches that even a single basepair mismatch significantly alters fluorescence (p. 60, lines 30-35), demonstrating probe selection based on stringent mismatch discrimination; the claimed no more than three basepairs represents an inherent property of the disclosed hybridization and alignment criteria (see MPEP 2112 for inherency).
Further, the Applicant’s argument that Ross fails to teach the constellation of method steps is also unpersuasive. Ross teaches sequence alignment, ranking/scoring, probe selection based on hybridization performance, and coupling to a solid matrix, as highlighted above. Therefore, showcasing the ordered steps recited in independent claim 1. The fact that Ross applies these techniques in neoplasm screening context does not negate anticipation, as intended use or field of application does not distinguish over a reference that discloses the same structural and procedural limitations (see MPEP 2111.02).
Accordingly, Ross discloses each and limitation of claims 1 and 7, either expressly or inherently, and the rejection under 102 is maintained.
Claim Rejections - 35 USC § 103
Claims 3-4 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (WO 2012/034170 A1, published 3/22/2012), as applied to claims 1 and 7 above, and in view of Redi et al. (“Genome Sizes in Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria”, Journal of Heredity; published 6/2005).
As discussed above, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that the DNA molecular or methylation array comprises a plurality of:
nucleic acid molecules comprising a nucleotide sequence corresponding to individualized DNA markers or a functional derivative, fragment, variant or homologue for either human or non-human mammalian genomes (p. 19, lines 1-10). Ross teaches that these libraries or polynucleotide sequence alignment sets were segregated to individual samples using the bar-code sequences and aligned with the bisulphite converted sequence of each amplicon, including the fraction of cytosines at each potential 5’-cytosine-phosphate-guanine-3’ (CpG) methylation site was determined for each sample—including homologous proteins (p. 30, lines 1-10; p. 88, lines 20-25) via the methylation of the overall amplicon or individual CpG sites can then be assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis within non-human mammalian genomes (p. 45, lines 20-25).
Regarding claims 3 and 4, Ross teaches that individual probes are composed of specified ranking criteria, representing the raw probe response value for the given combination of methylation status corresponding to methylation values in individual samples (p. 80, lines 5-10). Further, Ross teaches that gene-specific primers or individualized adapters are added to the ends of the randomly fragmented human DNA and the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, and further amplified using primers that cross-hybridize to the adaptor sequences within genomes of non-human mammalian species (p. 46, lines 10-15).
Ross does not teach or suggest that the previously described ranking comparisons applies to the specific homologs, Laurasiatheria, Euarchontoglires, Xenartha and Afrotheria superordinal groups, or that the specified alignments apply to at least 10 non-human mammalian species.
Redi teaches that 15 Xenarthra, 24 Euarchontoglires (Rodentia), as well as 23 Laurasiatheria (22 Chiroptera and 1 species from Perissodactyla) were established for a comprehensive set of genome size measurements (Abstract). Redi further teaches that Xenarthra exhibited much larger genomes in comparison to Chiroptera, while the genomes of Euarchontoglires and Laurasiatheria were found being smaller than those of Afrotheria and Xenarthra (Abstract). Further, Redi teaches that following evaluation of the previously mentioned genomes, only the genome sequence of the house mouse, appears in databases, while the Xenarthra are represented by only one species, the ant-eater, and the Afrotheria are represented just by the aardvark (Introduction: Paragraph 5). Further, Redi teaches that the placental classes were split into four phylogenetic classes; Afrotheria, and Xenarthra occupy basal positions, followed by the Boreoeutheria, which embrace Euarchontoglires and Laurasiatheria and showcased a shift toward smaller genomes during the transition of basal classes to the Boreoeutheria that conquered the Northern Hemisphere, following sequencing (Discussion: Paragraph 3). Redi also teaches that the nuclear DNA contents of 5 metatherian and 62 eutherian mammals were measured (Table 1; Results, Paragraph 1).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to modify Ross’ methodology of creating a DNA methylation array for sequence alignments of targeted genomes with Redi’s specific taxonomic groups, since both Ross and Redi are directed to analyzing genomic DNA from multiple mammalian species. While Ross teaches DNA methylation arrays that utilize sequence homology between human and non-human mammalian genomes, it lacks the specific taxonomic groups of Redi. However, Redi teaches detailed information on the phylogenetic relationships and genomic data across the specific mammalian superordinal groups (Laurasiatheria, Euarchontoglires, Xenartha and Afrotheria). One skilled in the art would recognize that Redi’s comprehensive mammalian taxonomic framework provides an organized approach to selecting which non-human mammalian species to include in Ross’ methylation array design. The combination would have a reasonable expectation of success because Ross already establishes the technical feasibility of creating DNA methylation arrays based on cross-species homology, providing the methodological foundation, since, Redi’s identification of specific mammalian taxonomic groups and their genomic relationships would expand Ross’ approach to include a more comprehensive set of mammalian species for improved cross-species applicability to encompass diverse mammalian lineages.
Applicant’s Response: The Applicant argues that the obviousness rejection fails because the cited references do not teach or suggest the specific methodological polynucleotide selection steps now recited in the amended claims. Specifically, the Applicant argues that the prior art does not teach selecting human genomic polynucleotide sequences that have no more than a three basepair mismatch with corresponding sequences in at least five non-human mammalian species, including both placental and non-placental mammals.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As set forth previously, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that this method includes performing sequence alignment, ranking/scoring probes, assessing CpG methylation following bisulfite conversion, and coupling nucleic acids to a solid matrix (p. 19, lines 1-10; p. 30, lines 1-10; p. 46, lines 10-15; p. 88, lines 20-25). Ross thus establishes both the technical feasibility and methodological framework for constructing cross-species methylation arrays based on conserved genomic sequences. However, Ross does not teach the specific mammalian superordinal groups recited in the dependent claims.
Redi teaches comprehensive genomic data and phylogenetic relationships across the specific mammalian superorders, and provides an organized taxonomic framework identifying representative species within those groups (Introduction: Paragraph 5; Table 1; Results, Paragraph 1). It would have been obvious to one of ordinary skill in the art to apply Redi’s well-established mammalian phylogenetic classifications to Ross’ cross-species methylation array methodology in order to select representative non-human mammalian species scanning divergent evolutionary lineages (see MPEP 2143). The motivation arises from the express goal of cross-species applicability and comparative genomic analysis, which both Ross and Redi address.
The Applicant’s argument that the references fail to teach the specific limitation of selecting sequences having no more than a three basepair mismatch across at least five non-human mammalian species is not persuasive. Ross teaches alignment, ranking, hybridization discrimination, and sensitivity to single-base mismatches (p. 60, lines 30-35), demonstrating probe selection based on stringent mismatch criteria. Once Ross establishes probe selection based on homology and hybridization performance, optimizing the allowable mismatch threshold (i.e., <3 basepairs) across multiple species represents routine parameter optimization, which is ordinarily obvious. Moreover, extending Ross’ cross-species approach to at least five species spanning placental and non-placental mammals is a predictable expansion of the comparative framework in view of Redi’s phylogenetic classifications.
The combination would have had a reasonable expectation of success because Ross already demonstrates that conserved sequences can be identified and used in methylation arrays, and Redi supplies the taxonomic structure for selecting diverse mammalian representatives. The claimed invention therefore represents the predictable use of prior art elements according to their established functions, and the rejection under 103 is maintained.
Claims 5-6 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (WO 2012/034170 A1, published 3/22/2012), as applied to claims 1 and 7 above, and in view of Irizarry et al. (WO 2009/021141 Al, published 2/12/2009, from IDS 7/8/2021).
As discussed above, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that the DNA molecular or methylation array comprises a plurality of:
nucleic acid molecules comprising a nucleotide sequence corresponding to individualized DNA markers or a functional derivative, fragment, variant or homologue for either human or non-human mammalian genomes (p. 19, lines 1-10). Ross teaches that these libraries or polynucleotide sequence alignment sets were segregated to individual samples using the bar-code sequences and aligned with the bisulphite converted sequence of each amplicon, including the fraction of cytosines at each potential 5’-cytosine-phosphate-guanine-3’ (CpG) methylation site was determined for each sample—including homologous proteins (p. 30, lines 1-10; p. 88, lines 20-25) via the methylation of the overall amplicon or individual CpG sites can then be assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis within non-human mammalian genomes (p. 45, lines 20-25).
Regarding claims 5-6, Ross teaches that targeted polynucleotides of interest isolated from a tissue of interest are hybridized to the DNA chip and the specific sequences detected based on the target polynucleotides' preference and degree of hybridization at discrete probe locations on the matrix (p. 72, lines 15-20).
Ross does not teach or suggest that the previously described DNA molecular or methylation array comprises at least 30,000 unique polynucleotides coupled to the matrix, or that the unique plurality of polynucleotides is between 40-80 nucleotides in length.
Irizarry teaches that the number of discrete genomic regions may vary, dependent on the parameters used for defining the regions (i.e., the size and type of the genome utilized), ranging from about 10, 50, 100, 1,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 100,000 genomic regions or unique polynucleotides targeted to be represented by probes on the array (Paragraph 48, lines 1-10). Further, Irizarry teaches that each previously described probe includes a sequence determined by tiling each discrete genomic region, with different sizes of oligonucleotide sequences ranging from about 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95 or 95-100 base pairs in length (Paragraph 50, lines 1-10).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to modify Ross’ methodology of creating a DNA methylation array for sequence alignments of targeted genomes with Irizarry’s specific guidance on array design parameters, since both references are directed to DNA methylation arrays and their design. While Ross teaches a DNA methylation array comprising polynucleotides coupled to a matrix for detecting patterns in both human and non-human genomes, Irizarry complements Ross by teaching specific guidance on array design parameters, including the number of discrete genomic regions and the specified lengths of polynucleotide sequences. One skilled in the art would have been motivated to apply Irizarry’s specific array design parameters to Ross’ methylation array to enhance its comprehensive genomic coverage and detection sensitivity, particularly since Irizarry identifies 30,000 as a specifically contemplated number of genomic regions for array representation. There would have been a reasonable expectation of success in combining these references because Ross already establishes the fundamental methodology for creating DNA methylation arrays, while Irizarry provides specific technical parameters that could be readily incorporated into Ross’ design without requiring undue experimentation, since the array density ranges and polynucleotide length ranges taught by Irizarry fall within conventional array manufacturing capabilities that existed at the time of the invention to yield improved resolution and coverage for detecting methylation patterns across species.
Applicant’s Response: The Applicant argues that the obviousness rejection fails because the cited references do not teach or suggest the specific methodological polynucleotide selection steps now recited in the amended claims. Specifically, the Applicant argues that the prior art does not teach selecting human genomic polynucleotide sequences that have no more than a three basepair mismatch with corresponding sequences in at least five non-human mammalian species, including both placental and non-placental mammals.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As set forth previously, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that this method includes performing sequence alignment, ranking/scoring probes, assessing CpG methylation following bisulfite conversion, and coupling nucleic acids to a solid matrix (p. 19, lines 1-10; p. 30, lines 1-10; p. 46, lines 10-15; p. 88, lines 20-25). Ross thus establishes both the technical feasibility and methodological framework for constructing cross-species methylation arrays based on conserved genomic sequences. However, Ross does not teach the recited array density (at least 30,000 unique polynucleotides) or the specific probe length range (40-80 nucleotides).
Irizarry teaches that DNA methylation arrays may comprise varying numbers of discrete genomic regions, including 30,000, 40,000, 50,000 or more unique polynucleotides represented by probes (Paragraph 48), and further teaches oligonucleotide probe lengths ranging from about 15 to 100 basepairs, including ranges that encompass 40-80 nucleotides (Paragraph 50). It would have been obvious to one of ordinary skill in the art to apply Irizarry’s specific array design parameters to Ross’ methylation array methodology in order to increase genomic coverage, resolution, and detection sensitivity. Combining known design parameters with a known array platform to achieve predictable improvements constitutes a proper rationale under MPEP 2143 and represents routine optimization of result-effective variables under MPEP 2144.05.
The Applicant’s argument that the references fail to teach the specific limitation of selecting sequences having no more than a three basepair mismatch across at least five non-human mammalian species is not persuasive. Ross teaches alignment, ranking, hybridization discrimination, and sensitivity to single-base mismatches (p. 60, lines 30-35), demonstrating probe selection based on stringent mismatch criteria. Once Ross establishes probe selection based on homology and hybridization performance, optimizing the allowable mismatch threshold (i.e., <3 basepairs) across multiple species represents routine parameter optimization, which is ordinarily obvious, as set forth previously. Claims 5-6 further limit only the number and length of probes on the array, Irizarry teaches these quantitative and dimensional parameters. The combination does not change Ross’ selection methodology, but merely applies well-known array density and probe length design choices to it.
A person of ordinary skill in the art would have had a reasonable expectation of success because Ross provides the foundational cross-species methylation array framework, and Irizarry provides conventional and expressly disclosed array density and probe length parameters that fall within established manufacturing capabilities (see MPEP 2143, 2144.05). The claimed subject matter therefore represents the predictable use of prior art elements according to their established functions, and the rejection of claims 5-6 under 103 is maintained.
Claims 8-11 are rejected under 35 U.S.C. 103 as being unpatentable over Ross et al. (WO 2012/034170 A1, published 3/22/2012), as applied to claims 1 and 7 above, in view of Irizarry et al. (WO 2009/021141 Al, published 2/12/2009, from IDS 7/8/2021), and in further view of Redi et al. (“Genome Sizes in Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria”, Journal of Heredity; published 6/2005) and in further view of Janke et al. (“Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, Vombatus ursinus, and the Spiny Anteater, Tachyglossus aculeatus: Increased Support for the Marsupionta Hypothesis”, J Mol Evol.; published 2002).
As discussed above, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that the DNA molecular or methylation array comprises a plurality of:
nucleic acid molecules comprising a nucleotide sequence corresponding to individualized DNA markers or a functional derivative, fragment, variant or homologue for either human or non-human mammalian genomes (p. 19, lines 1-10).
Regarding claims 8-11, Ross teaches that the DNA molecular or methylation array described previously comprises DNA variants or homologues for either human or non-human mammalian genomes (p. 19, lines 1-10). Ross also teaches a polynucleotide sequence alignment for scoring, specifically via blood DNA sample (i.e., human or non-human) were separately ligated with bar coded "MID" linkers for later scoring or ranking (p. 23, lines 25-30; p. 88, lines 15-20). Ross teaches that these libraries or polynucleotide sequence alignment sets were segregated to individual samples using the bar-code sequences and aligned with the bisulphite converted sequence of each amplicon, including the fraction of cytosines at each potential 5’-cytosine-phosphate-guanine-3’ (CpG) methylation site was determined for each sample—including homologous proteins (p. 30, lines 1-10; p. 88, lines 20-25) via the methylation of the overall amplicon or individual CpG sites can then be assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis within non-human mammalian genomes (p. 45, lines 20-25). Ross also teaches that individual probes are composed of specified ranking criteria, representing the raw probe response value for the given combination of methylation status corresponding to methylation values in individual samples (p. 80, lines 5-10). Further, Ross teaches that gene-specific primers or individualized adapters are added to the ends of the randomly fragmented human DNA and the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, and further amplified using primers that cross-hybridize to the adaptor sequences within genomes of non-human mammalian species (p. 46, lines 10-15). Ross further teaches that the previously described individualized probes for ranking permit distinction between the ligated and unligated probes, in which case the presence of both labels in the same eluate fraction confirms the ligation event and the target nucleic acid is bound to a solid matrix (p. 58, lines 5-10).
Ross also teaches that the previously described DNA methylation array is composed of polynucleotides from human genomes that even a single base pair mismatch compared to a non-human mammalian genome significantly alters the level of fluorescence detected in a sample (p. 60, lines 30-35).
Ross teaches that the previously described molecular or methylation arrays are also useful for differentiating between a mutated and non-mutated sequence (p. 58, lines 10-15).
Additionally, Ross teaches that the non-human mammals include primates, livestock animals (i.e., horses, cattle, sheep, pigs, donkeys), laboratory test animals (i.e., mice, rats, rabbits, guinea pigs), companion animals (i.e., dogs, cats) and captive wild animals (p. 23, lines 30-35).
Ross does not teach or suggest that the previously described ranking comparisons applies to a 60-nucleotide segment in specific homologs, Laurasiatheria, Euarchontoglires, Xenartha and Afrotheria superordinal groups, or monotreme or marsupial mammalian species, as part of at least 2,000 unique polynucleotide sequences. Further, Ross does not teach or suggest that at least 2,000 polynucleotides within the plurality of polynucleotide sequences can hybridize to a 40-nucleotide segment in genomic polynucleotide sequences of a marsupial, monotreme, Laurasiatheria, Euarchontoglires, Xenartha and Afrotheria mammalian species to comprise a DNA methylation array. Further, Ross does not teach or suggest specified species for the mammalian species; including the marsupial mammalian species is a Wallaby species; the monotreme mammalian species is a Platypus species; the Xenarthra mammalian species is an armadillo species; the Laurasiatheria mammalian species is a bat species and/or the Afrotheria mammalian species is a tenrec species.
Irizarry teaches that the number of discrete genomic regions may vary, dependent on the parameters used for defining the regions (i.e., the size and type of the genome utilized), ranging from about 10, 50, 100, 1,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 100,000 genomic regions or unique polynucleotides targeted to be represented by probes on the array (Paragraph 48, lines 1-10). Further, Irizarry teaches that each previously described probe includes a sequence determined by tiling each discrete genomic region, with different sizes of oligonucleotide sequences ranging from about 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95 or 95-100 base pairs in length (Paragraph 50, lines 1-10).
Redi teaches that 15 Xenarthra, 24 Euarchontoglires (Rodentia), as well as 23 Laurasiatheria (22 Chiroptera and 1 species from Perissodactyla) were established for a comprehensive set of genome size measurements (Abstract). Redi further teaches that Xenarthra exhibited much larger genomes in comparison to Chiroptera, while the genomes of Euarchontoglires and Laurasiatheria (included 50 bat species) were found being smaller than those of Afrotheria (i.e., elephants, hyraxes, aardvarks and tenrecs) and Xenarthra (Abstract; Results: Paragraphs 3-5). Further, Redi teaches that following evaluation of the previously mentioned genomes, only the genome sequence of the house mouse, appears in databases, while the Xenarthra (included 12 species of armadillos) are represented by only one species, the ant-eater, and the Afrotheria are represented just by the aardvark (Introduction: Paragraphs 3-5). Further, Redi teaches that the placental classes were split into four phylogenetic classes; Afrotheria, and Xenarthra occupy basal positions, followed by the Boreoeutheria, which embrace Euarchontoglires and Laurasiatheria and showcased a shift toward smaller genomes during the transition of basal classes to the Boreoeutheria that conquered the Northern Hemisphere, following sequencing (Discussion: Paragraph 3). Redi also teaches that the nuclear DNA contents of 5 metatherian and 62 eutherian mammals were measured (Table 1; Results, Paragraph 1).
Janke teaches that the monotremes; the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to
the common belief that they represent the living survivors of an ancestral stock of mammals, however, revealing analyses showcased data from the complete mitochondrial (mt) genomes of a
second monotreme, the spiny anteater, and another marsupial, the wallaby, yielded clear support for the Marsupionta hypothesis contrary to the conventional view that marsupials and placentals form a clade (Theria) that excludes monotremes (Abstract).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to modify Ross’ methodology of creating a DNA methylation array for sequence alignments of targeted genomes with Irizarry’s specific array design parameters, including the number of genomic regions and oligonucleotide sequence lengths, Redi’s critical taxonomic information on mammalian evolutionary relationships, detailing the genomic characteristics of specified ordinal groups for cross-hybridization studies, and Janke’s insights into monotremes and marsupials that would enable inclusion of these evolutionary distinct mammals in comprehensive methylation array designs. A person of ordinary skill in the art would have been motivated to combine these references to create a more comprehensive DNA methylation array with improved cross-species applicability. Further, Ross teaches the applications of the methylation arrays to non-human mammalian species, while Redi, Irizarry, and Janke collectively provide the specific tools needed to optimize such arrays by incorporating the full diversity of mammalian taxonomic groups. Additionally, Ross teaches that even a single base mismatch impacts detection capabilities (p. 60, lines 30-35); therefore, incorporation of specific taxonomic insights, like those from Redi and Janke, would provide appropriate cross-hybridization properties.
There would have been a reasonable expectation of success in combining these teachings because Ross already established the technical feasibility of creating DNA methylation arrays for cross-species applications. The additional parameters from Irizarry regarding array design, coupled with the specific mammalian genomic information from Redi and Janke, would enable one of ordinary skill in the art, to implement these improvements using conventional array manufacturing techniques without undue experimentation.
Applicant’s Response: The Applicant argues that the obviousness rejection fails because the cited references do not teach or suggest the specific methodological polynucleotide selection steps now recited in the amended claims. Specifically, the Applicant argues that the prior art does not teach selecting human genomic polynucleotide sequences that have no more than a three basepair mismatch with corresponding sequences in at least five non-human mammalian species, including both placental and non-placental mammals.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered but are not found persuasive, as discussed below.
As set forth previously, Ross teaches a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules via a DNA molecular or methylation array (Abstract). Further, Ross teaches that this method includes performing sequence alignment, ranking/scoring probes, assessing CpG methylation following bisulfite conversion, and coupling nucleic acids to a solid matrix (p. 19, lines 1-10; p. 30, lines 1-10; p. 46, lines 10-15; p. 88, lines 20-25). Ross thus establishes both the technical feasibility and methodological framework for constructing cross-species methylation arrays based on conserved genomic sequences. Further, Ros teaches that even a single base mismatch can affect hybridization detection (p. 60, lines 30-25), demonstrating the use of stringent sequence homology when designing probes for cross-species methylation analysis.
Irizarry teaches array design parameters including the number of genomic regions represented on a methylation array and probe lengths used in array construction, specifically teaching arrays comprising up to tens of thousands of genomic regions and probes having lengths ranging from about 15 to 100 base pairs, including ranges encompassing the claimed probe lengths (Paragraphs 48-50). Further, Redi teaches phylogenetic relationships and genome characteristics across mammalian superordinal groups providing an established taxonomic framework for selecting representative mammalian species for genomic comparison (Abstract; Results: Paragraphs 3-5). Janke further teaches evolutionary relationships involving monotremes and marsupials, thereby providing additional guidance regarding inclusion of evolutionary distant mammalian lineages (Abstract).
It would have been obvious to one of ordinary skill in the art to modify Ross’ cross-species DNA methylation array methodology by incorporating Irizarry’s well-known array design parameters (i.e., probe number and probe length ranges) and by selecting representative mammalian species spanning diverse evolutionary lineages as taught by Redi and Janke. Such a combination would allow construction of a methylation array capable of detecting methylation patterns across a broad phylogenetic range of mammalian species. Combining prior art elements according to known methods to yield predictable results constitutes a proper rationale for obviousness (see MPEP 2143). Furthermore, selecting probe number, probe length, and species coverage represents routine optimization of result-effective variables in array design (see MPEP 2144.05).
Although the Applicant argues that the cited references fail to teach or suggest the specific methodological polynucleotide selection step requiring sequences having no more than a three basepair mismatch across at least five non-human mammalian species including placental and non-placental mammals, this argument is not persuasive. Ross already teaches sequence alignment and hybridization discrimination based on sequence mismatches and homology between human and non-human genomes (p. 23, lines 25-30; p. 60, lines 30-25), thereby establishing the principle of selecting probes based on cross-species sequence similarity. Extending Ross’ cross-species approach to multiple representative mammalian lineages as taught by Redi and Janke would have been an obvious design choice for improving cross-species applicability of the methylation array. The particular mismatch threshold represents an optimization of hybridization parameters and would have been within the routine skill of the art (see MPEP 2144.05).
Therefore, a person of ordinary skill in the art would have had a reasonable expectation of success because Ross establishes the technical feasibility of cross-species DNA methylation arrays, while Irizarry provides conventional array construction parameters and Redi and Janke provide well-known phylogenetic frameworks identifying representative mammalian species. The claimed invention therefore represents the predictable use of known techniques and parameters for designing methylation arrays capable of analyzing diverse mammalian genomes. Accordingly, the rejection of claims 8-11 under 103 is maintained.
Allowable Subject Matter
Regarding claim 12, although the primary reference (Ross, as cited above to anticipate claims 1 and 7, and used in view of Irizarry, Redi and Janke for claims 3-6 and 8-11) teaches methods for screening modulation in DNA methylation using DNA methylation arrays comprising polynucleotides corresponding to individualized DNA markers or homologues for human or non-human mammalian genomes (p. 19, lines 1-10; p. 30, lines 1-10; p. 45, lines 20-25), Ross does not teach or suggest the specific polynucleotide sequence shown in SEQ ID NO: 1 within the plurality of polynucleotides recited in the presently claimed array.
Specifically, Ross teaches the use of polynucleotides corresponding to genomic markers or homologous sequences for methylation detection, but does not disclose or suggest the particular sequence recited in SEQ ID NO: 1, as required by instant claim 12. Further, Hatchwell et al. (US PGPub 2018/0223360 A1; published 8/9/2018) does not set forth the exact sequence, with no additional basepairs, as required by instant claim 12. Hatchwell teaches the sequence of SEQ ID NO: 1 with 100% similarity, however the sequence has a length of 303039 (SEQ ID NO: 1913). Therefore, neither Ross, nor in combination with Irizarry, Redi, Janke or Hatchwell, teach or suggest incorporating the specific sequence shown in SEQ ID NO: 1 into the DNA methylation array as claimed.
Therefore, claim 12 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the based claim and intervening claims.
Conclusions
No claim is allowed.
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 extension fee 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 date of this final action.
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684