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 .
This Office Action is in reply to Applicants’ correspondence of 5/6/2026. Applicants’ remarks and amendments have been fully and carefully considered but are not found to be sufficient to put this application in condition for allowance. New grounds of rejection, necessitated by amendments, are presented in this Office Action. Any rejections or objections not reiterated herein have been withdrawn in light of the amendments to the claims or as discussed in this Office Action. This Action is FINAL.
Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Status
Claims 53 and 56-80 are pending and being examined on the merits.
Nucleotide and/or Amino Acid Sequence Disclosures
Applicant’s submission of replacement drawings for figures 19, 22, 23, 26, 29, and 30 with appropriate identification of sequences with SEQ ID NOs is acknowledged. Additionally, Applicant’s submission of a new Sequence Listing and amendment to the specification to reflect this change is acknowledged.
Specification
The objection to the specification is withdrawn in light of Applicant’s amendment to the specification to remove the references to SEQ ID NOs in paragraphs [0020-0023] (opting instead to include the appropriate SEQ ID NOs in the drawings themselves).
Applicant’s amendment of the specification to properly denote trade names/marks used in commerce is acknowledged.
Claim Objections
Withdrawn: The objection of claims 53, 62, and 74 as detailed in the Office Action of 2/6/2026 is withdrawn in light of Applicant’s amendments to the claims.
New (Necessitated by Amendments): Claims 57, 63, and 75 are objected to because of the following informalities:
Claim 57 reads “computing, for each target, the ratio between the respective target peak and the respective spike-in peak intensity” and should read “computing, for each target, the ratio between the respective target peak intensity and the respective spike-in peak intensity”.
Claims 63 and 75 read “determining the presence or absence of an aneuploidy based on the computed ratios” and should read “determining the presence or absence of an aneuploidy based on the computed chromosome-specific ratios” to maintain consistent claim terminology with the newly amended claims 62 and 74.
Appropriate correction is required.
Claim Rejections - 35 USC § 112b - Indefiniteness
Withdrawn: The rejection of claims 53-78 under 35 U.S.C. 112(b) as detailed in the Office Action of 2/6/2026 is withdrawn in light of Applicant’s amendments to the claims.
New (Necessitated by Amendments):
Claims 53 and 56-80 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 53 recites the limitation “wherein each of the plurality of spike-in molecules further comprises: a target region having a first nucleotide sequence with sequence similarity to a target sequence region; a variation region having a nucleotide sequence with sequence dissimilarity to the target sequence”. This does not impart adequate structural information regarding the sequence of the spike-in molecules. It is unclear how the spike-in molecule amplicon peak would be differentiated from its associated endogenous target amplicon peak from claim language alone. This is especially unclear given that no information is provided as to how peak data is calculated before being received (i.e., no information regarding the methodology to generate said peak data is provided). Further clarification is required.
Claims 56-80 depend from claim 53, inherit this deficiency, and are rejected on the same basis.
Claims 56 and 57 are indefinite as being both incomplete, by their dependence on a cancelled claim, and for lack of antecedent basis for their limitations which are no longer present due to the cancellation of claim 55. For the purposes of examination, these claims are being interpreted as depending from claim 53, however correction is required.
Response to Remarks
In Applicant’s Response of 5/6/2026, they explain that it is clear from the specification that the target-specific primers amplify both the endogenous target and the target-associated spike-in molecule (paragraph [0082] of US 20220340971 A1, for example). This argument is found persuasive and the relevant rejection to claim 53 has been withdrawn. Given applicant’s subsequent argument that the same applies for the fluorescently labeled primers (i.e., they bind to the same sequence which is shared between the endogenous target and the associated spike-in molecule) and the explanation of the use of said primers in the specification, the relevant rejection against the fluorescent primers is withdrawn as well.
Applicant did not address the 112b indefiniteness rejection against claim 53 regarding the lack of adequate structural information of the spike-in molecules. The examiner acknowledged that the spike-in molecule must have regions of similarity to the associated target molecule in order to be amplified and labeled by the same primer sets. However, the “variation” sequence is indefinite in that it is unclear how this variation makes the spike-in molecule distinguishable from the target molecule. This is especially unclear given that the downstream process used to generate peak intensities is not specified in the claims and thus does not impart any structural limitation on the variation (i.e., does not indicate what would make the variation region dissimilar in a way that would allow for the peak intensities to be distinguishable).
Claim Rejections - 35 USC § 112d – Failure to Further Limit
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 67 and 68 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. In the instant case, claims 67 and 68 fail to further limit claim 53, from which they depend. All limitations of claims 67 and 68 are present in independent claim 53 in the most recent set of claim amendments (submitted 5/6/2026). Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
Withdrawn:
The claim rejections under 35 U.S.C. 103 as detailed in the Office Action of 2/6/2026 are withdrawn in light of Applicant’s amendments to the claims.
New (Necessitated by Amendments):
Claims 53, 56-62, 64-74, and 76-80 are rejected under 35 U.S.C. 103 as being unpatentable over Tsao (Tsao et al., US 2019/0095577 A1; cited on IDS of 8/2/2023) in view of Holck (Holck et al., European Food Research and Technology 2011; cited on PTO-892 of 2/6/2026) and Nygren (US 2012/0264618 A1; cited on PTO-892 of 2/6/2026).
As noted in the 112b rejection above, claims 56 and 57 depend from a cancelled claim (claim 55). For the purposes of examination, these claims are being interpreted as depending from claim 53.
Claims 53, 67, and 68: Tsao teaches a method of determining abundance metrics of endogenous targets through computing ratios between the amplicons of endogenous targets and amplicons of target-associated molecules (reads on spike-in molecules; Abstract and paragraph [0018]). Tsao teaches mixing a nucleic acid sample of a subject and a plurality of spike-in molecules to create a mixture, each of the plurality of spike-in molecules are associated with an endogenous target or targets of a set of targets (paragraph [0023]). Tsao teaches that the plurality of spike-in molecules further comprise a target region having a first nucleotide sequence with sequence similarity to a target sequence region and a variation region having a second nucleotide sequence with sequence dissimilarity to the target sequence region (paragraph [0023]). Tsao teaches co-amplifying the mixture with target-specific primers to create a co-amplified mixture (paragraph [0023]). Tsao teaches receiving peak data from the co-amplified mixture, the peak data including, for each target of the set of targets, peak intensities of the nucleic acid sample and spike-in speak intensities of the spike-in molecules associated with each respective target (paragraph [0023]). Tsao teaches, for each target, combining the peak intensities of the nucleic acid and combining the spike-in peak intensities (paragraph [0023]) and for each target, computing a ratio between the respective average target peak intensity and the respective average spike-in peak intensity and determining the abundance of the target based on computed ratios (paragraph [0023]). Tsao teaches that the sample is a DNA sample (“endogenous DNA molecules…from a sample (e.g., a maternal blood sample)”). Tsao teaches that the target of the set of targets comprises a chromosome of a set of chromosomes and the target-specific primers are chromosome-specific primers that are each configured to capture a respective chromosome in the set of chromosomes (paragraph [0023]). Tsao teaches that combining the peak intensities of the nucleic acid for a particular chromosome target comprises combining peak intensities for different fragment of the particular chromosome target (“where the different target sequence regions or the target sequence can correspond to different loci”, “e.g., averaging individual abundance ratios for different pairs of target sequence base and target-associated sequence base associated with a plurality of target loci for chromosome 21; averaging abundance ratios across loci; etc.” paragraph [0023, 0046]). Tsao teaches determining the abundance of the target comprises determining the presence or absence of an aneuploidy based on the computed ratios for two or more chromosomes (paragraph [0023], comparison of the abundance ratio for chromosome 21 to the abundance ratio of a reference chromosome such as chromosome 18).
Tsao does not teach labeling the co-amplified mixture by fluorescently labeled primers. However, the use of fluorescently labeled primers to label co-amplified mixtures for subsequent detection and determination of relative abundances is known in the art, as taught by Holck.
Holck teaches a multiplex quantitative PCR method in which target molecules are co-amplified with synthetic competitor molecules (reads on spike-in molecules or “target-associated molecules”; Abstract and Table 3). Holck teaches amplifying the target molecules and the target-associated molecules using this primer set to generate a labeled, co-amplified mixture.
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Tsao, who teaches co-amplification of targets and target-associated molecules, to include fluorescently labeled primers for co-amplification to generate a labeled co-amplification mixture, as taught by Holck. One would be motivated to do so given that Holck teaches that inclusion of fluorescent primers allows the differentiation of fragments through capillary electrophoresis and couples it with fluorescent detection for “high resolution of fragments, high sensitivity and high-throughput capabilities” and “easy identification of amplicons even when the sizes of PCR fragments are almost identical”. One would have a reasonable expectation of success given that Tsao teaches that their method is compatible with capillary electrophoresis (paragraph [0068]). Additionally, Holck teaches that any qualitative PCR approach can easily incorporate the fluorescently labeled primers without changing the primer sets themselves and requires no additional steps be added to the traditional qualitative PCR methodologies (pg 952, col 1 last paragraph into col 2 first paragraph).
Tsao in view of Holck do not teach co-amplifying chromosome-specific targets and spike-in molecules with chromosome-specific primers that have tails to generate amplicons of discrete lengths. However, use of tailed primers for generating amplicons of a discrete length of a set of discrete lengths is known in the art, as taught by Nygren.
Nygren teaches a method of determining the relative amount of a nucleic acid species in a sample (Abstract). Nygren teaches using “competitive PCR” to co-amplify a target and target-associated molecules with the same primer pair, and then using capillary electrophoresis to separate the fragments and determine relative abundance through computation of a ratio between the two (paragraphs [0020, 0106, 0259] and Figure 1). Nygren teaches that “amplified competitors can optionally be further distinguishable from one another through the use of tailed amplification primers” (paragraph [0112]) and that in cases when multiple genomic targets are being amplified in the same reaction “the primers that amplify the genomic DNA target sequences and corresponding competitor oligonucleotides each contain a 5’ tail of the same overall length” (paragraph [0165]). Nygren teaches that “In a multiplex amplification assay, for example, where a plurality of genomic DNA target sequences are co-amplified with a plurality of corresponding competitor oligonucleotides, the genomic DNA target sequences can all be of the same length, and the competitor oligonucleotides can be of the same or different lengths, such that the amplicons generated (through the use of tailed primers of varying length, for example) are distinct for each region assayed. In such cases, each region would be represented by two amplicon lengths (i.e. one amplicon length for the genomic DNA target(s) for that region and another amplicon length for the corresponding competitor oligonucleotide(s))” (paragraph [0184]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Tsao in view of Holck, who teach co-amplification of chromosome targets and target-associated molecules using labeled primers, to include tailed primers for co-amplification to generate discrete fragment lengths for both target and target-associated molecules for a given region, as taught by Nygren. One would be motivated to do so given that Nygren teaches that amplifying fragments of the same length and then varying the length with tailed primers allows for the removal of “unnecessary amplification bias” (paragraph [0260]). One would have a reasonable expectation of success given that the methodology of Tsao in view of Holck already employs fragment separation via capillary electrophoresis (thus the length of the amplicons is being used to generate peak data), and the incorporation of the tailed primers for greater control of final amplicon length independent of the length of the target genomic region provides an additional level of controllable multiplex ability.
Claims 56 and 69: Tsao in view of Holck and Nygren teach computing, for each discrete length of the set of discrete lengths, a ratio between the respective target peak intensity and the spike-in peak intensity and then aggregating these individual abundance ratios across each discrete length of the set of discrete lengths (“for each pair, extracting an individual abundance ratio of endogenous to spike-in based on comparing a peak intensity metric…[and] generating an overall abundance ratio based on the individual abundance ratios (e.g., averaging individual abundance ratios for different pairs of target sequence base and target-associated sequence base associated with a plurality of target loci for chromosome 21; averaging abundance ratios across loci; etc.)” Tsao, paragraph [0023]).
Claims 57 and 70: Tsao in view of Holck and Nygren teach computing, for each discrete length of the set of discrete lengths, a ratio between the respective target peak intensity and the spike-in peak intensity and then aggregating these individual abundance ratios across each discrete length of the set of discrete lengths (“for each pair, extracting an individual abundance ratio of endogenous to spike-in based on comparing a peak intensity metric…[and] generating an overall abundance ratio based on the individual abundance ratios (e.g., averaging individual abundance ratios for different pairs of target sequence base and target-associated sequence base associated with a plurality of target loci for chromosome 21; averaging abundance ratios across loci; etc.)” Tsao, paragraph [0023]). Tsao in view of Holck and Nygren do not teach first aggregating the target peak intensity across each discrete length of the set of discrete lengths and aggregating the spike-in peak intensity across each discrete length of the set of discrete lengths and then computing a ratio between the aggregated target peak intensity and aggregated spike-in peak intensities. However, with respect to the order of steps, it is noted that the courts have held that any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C. Therefore, the claimed order of steps (aggregating prior to determining the ratio vs aggregating after determining individual ratios) is an obvious variant of the steps of the cited prior art.
Claims 58-59 and 71-72: Tsao teaches that the variation region includes an insertion of base pairs with a length of 1 base pair. Tsao also teaches that the variation region includes a deletion of base pairs with a length of 1 base pairs (“variation regions with one or more insertions…or one or more deletions”, paragraph [0078]). Tsao specifically exemplifies a variation region that is a deletion of 3 base pairs (paragraph [0079]).
Claims 60 and 73: Tsao teaches a respective variation region of a spike-in molecule is in the center of a respective amplicon of the spike-in molecule (Figures 2 and 3).
Claim 61: Holck teaches that each of the fluorescently labeled primers is associated with a color channel (Figure 2 and Table 3).
Claim 62: Tsao teaches that the sample is a DNA sample and that the target comprises a chromosome of a set of chromosomes (Tsao, paragraph [0023]). As described in the rejection of claim 53 above, Nygren teaches performing co-amplification with primers that add a tail with a discrete length of a set of discrete lengths to an amplicon of the DNA sample and a tail with the discrete length of the set of discrete lengths to an amplicon of the spike-in molecule. The rationale to combine said references is provided in the rejection of claim 53 above. Tsao teaches aggregating the peak intensity ratios (discrete length-specific ratios) across each of the discrete lengths in the set of discrete lengths to generate a chromosome-specific ratio (Tsao, paragraph [0023]; see also the rejection of claims 56 and 69 above). Tsao teaches determining the abundance of the target comprises determining the presence or absence of an aneuploidy based on the computed chromosome-specific ratios for two or more chromosomes (paragraph [0023]).
Claim 64 and 65: Tsao teaches that the variation region includes an insertion of base pairs with a length of 1 base pair. Tsao also teaches that the variation region includes a deletion of base pairs with a length of 1 base pairs (“variation regions with one or more insertions…or one or more deletions”, paragraph [0078]). Tsao specifically exemplifies a variation region that is a deletion of 3 base pairs (paragraph [0079]).
Claim 66: Tsao teaches a respective variation region of a spike-in molecule is in the center of a respective amplicon of the spike-in molecule (Figures 2 and 3).
Claim 74: Tsao teaches that the sample is a DNA sample and that the target comprises a chromosome of a set of chromosomes (Tsao, paragraph [0023]). As described in the rejection of claim 53 above, Nygren teaches performing co-amplification with primers that add a tail with a discrete length of a set of discrete lengths to an amplicon of the DNA sample and a tail with the discrete length of the set of discrete lengths to an amplicon of the spike-in molecule. Nygren teaches that the addition of a tail using a tailed primer can be done to a subset of amplicons (Nygren, Figure 1 molecule 1). The rationale to combine said references is provided in the rejection of claim 53 above. Tsao teaches aggregating the peak intensity ratios (discrete length-specific ratios) across each of the discrete lengths in the set of discrete lengths to generate a chromosome-specific ratio (Tsao, paragraph [0023]; see also the rejection of claims 56 and 69 above). Tsao teaches determining the abundance of the target comprises determining the presence or absence of an aneuploidy based on the computed chromosome-specific ratios for two or more chromosomes (paragraph [0023]).
Claim 76 and 77: Tsao teaches that the variation region includes an insertion of base pairs with a length of 1 base pair. Tsao also teaches that the variation region includes a deletion of base pairs with a length of 1 base pairs (“variation regions with one or more insertions…or one or more deletions”, paragraph [0078]). Tsao specifically exemplifies a variation region that is a deletion of 3 base pairs (paragraph [0079]).
Claim 78: Tsao teaches a respective variation region of a spike-in molecules is in the center of a respective amplicon of the spike-in molecule (Figures 2 and 3).
Claims 79-80: Tsao teaches averaging peak intensities from the different fragments of the particular chromosome target (paragraph [0045, 0085]). Averaging peak intensities necessarily requires first aggregating the peak intensities from the different fragments.
Claims 63 and 75 are rejected under 35 U.S.C. 103 as being unpatentable over Tsao (Tsao et al., US 2019/0095577 A1; cited on IDS of 8/2/2023) in view of Holck (Holck et al., European Food Research and Technology 2011; cited on PTO-892 of 2/6/2026) and Nygren (US 2012/0264618 A1; cited on PTO-892 of 2/6/2026) as applied to claims 53, 56-62, 64-74, and 76-80 above, and further in view of Landry (Landry et al., US 2019/0147980 A1; cited on IDS of 8/2/2023).
The teachings of Tsao in view of Holck and Nygren are detailed above. Relevant to the instantly rejected claims, Tsao in view of Holck and Nygren teach computing a ratio of abundance of a target chromosome relative to the abundance of a target-associated spike-in molecule and using the computed ratios relative to a reference chromosome ratio to determine an aneuploidy (such as Down Syndrome, see Tsao paragraph [0023]).
Tsao in view of Holck and Nygren do not teach determining that the computed ratio is greater than a threshold ratio to determine the presence of aneuploidy. However, comparison of a computed abundance ratio of target chromosomes to reference chromosomes and comparison of this ratio to a threshold to accurately diagnose an aneuploidy is known in the art, as taught by Landry.
Landry teaches a method wherein an endogenous DNA target (such as a region on chromosome 21) is co-amplified with target-associated spike-in molecules and a reference target (a chromosome other than chromosome 21) and reference target-associated spike-in molecules (which reads on the plurality of other chromosome-specific ratios; paragraph [0060]). Landry teaches determining a ratio between each target/target associated pair intensity and each reference target/reference target-associated pair intensity (paragraph [0060]). Landry then teaches computing a ratio between the target-associated ratio and the reference-associated ratio, and if the target-associated ratio relative to the reference-associated ratio exceeds a “statistically significant threshold amount”, diagnosing aneuploidy (paragraph [0060]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Tsao in view of Holck and Nygren, who teach comparison of target-associated ratios to reference-associated ratio for diagnosis of an aneuploidy, to include a threshold ratio as taught by Landry. One would be motivated to include a threshold ratio comparison given the teaching by Landry that this allows a “statistically significant” method of determining an aneuploidy based on ratio comparisons (paragraph [0060]). One would have a reasonable expectation of success given that Landry is using this threshold methodology in comparing ratios that are obtained through relative abundance ratios between targets and target-associated molecules, as taught by Tsao.
Response to Remarks
Applicant traverses the rejection of all claims under 35 U.S.C. 103 as detailed in the previous Office Action of 2/6/2026 (pages 14-16 of Remarks). Applicant's arguments filed 5/6/2026 have been fully considered but they are not persuasive for the following reasons.
Applicant argues on pages 14-15 of Remarks that the newly added amendments to claim 53 (averaging peak intensities, DNA sample, chromosomal targets, etc.) are not taught by the cited references. These additional limitations have been addressed by the new claim rejections presented above.
Applicant then argues against Tsao, Holck, and Nygren (page 15 of Remarks). In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues that the methodology of Nygren “distinguishes between signals from different sources” while the claimed methods require “combining signal from different sources”. However, these are not mutually exclusive. Nygren teaches generating chromatogram data (peak intensities) that are distinguishable by length (rather than by, say, color). Nygren is merely teaching an alternative methodology of distinguishing between molecule populations, while Tsao is teaching how said data is then processed (such as by aggregating specific peaks).
Applicant argues that “the use of fluorescently labeled primers necessitates only a single amplification and labeling step, thereby improving the efficiency of the method” (page 15 of Remarks). In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., only one set of primers) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). The current claims involve the use of “target-specific primers” to create the co-amplified mixture (line 11 of claim 53) and then in a separate step uses “one or more fluorescently labeled primers” to label the co-amplified mixture (line 12 of claim 53). As claimed, two different primer sets are being used.
Double Patenting
Withdrawn: The claim rejections under Double Patenting as detailed in the Office Action of 2/6/2026 are withdrawn in light of Applicant’s amendments to the claims. New claim rejections, necessitated by the amendments, are presented below.
New (Necessitated by Amendments):
US 11,430,543 B2
Claims 53 and 56-80 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 6, and 13 of U.S. Patent No. US 11,430,543 B2 in view of Holck (Holck et al., European Food Research and Technology 2011; cited on PTO-892 of 2/6/2026), Landry (Landry et al., US 2019/0147980 A1; cited on IDS of 8/2/2023), and Nygren (US 2012/0264618 A1; cited on PTO-892 of 2/6/2026).
Regarding claims 53, 67, and 68: ‘543 teaches determining abundance through generation of a plurality of spike-in molecules associated with an endogenous target wherein the spike-in molecules have a target region with sequence similarity to a target sequence region and a variation region having sequence dissimilarity to the target sequence region, co-amplifying the mixture, receiving peak data from the co-amplified mixture and determining a target-associated abundance metric based on the peak data (reads on computing a ratio). ‘543 does not teach labeling the co-amplified mixture with fluorescently labeled primers (claim 53) or performing the amplification with fluorescently labeled primers (claim 62). Additionally, ‘543 does not teach that the variation sequence is in the center of the spike-in molecule (claims 60 and 73). However, the use of fluorescently labeled primers to label co-amplified mixtures for subsequent detection and determination of relative abundances with variation regions in the center of the molecule is known in the art, as taught by Holck.
Holck teaches a multiplex quantitative PCR method in which target molecules are co-amplified with synthetic competitor molecules (reads on spike-in molecules or “target-associated molecules”; Abstract and Table 3). Holck teaches amplifying the target molecules and the target-associated molecules using this primer set to generate a labeled, co-amplified mixture. Holck teaches that each of the fluorescently labeled primers is associated with a color channel (Figure 2 and Table 3). Holck teaches that the synthetic competitor molecules have variation regions in the center of the molecules (Figure 1).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of ‘543, who teaches co-amplification of targets and target-associated molecules, to include fluorescently labeled primers for co-amplification to generate a labeled co-amplification mixture, as taught by Holck. One would be motivated to do so given that Holck teaches inclusion of fluorescent primers allows the differentiation of fragments with fluorescent detection for “high resolution of fragments, high sensitivity and high-throughput capabilities” and “easy identification of amplicons even when the sizes of PCR fragments are almost identical”. One would be motivated to have the variation region towards the center of the molecule given the teaching by Holck that this allows for the variation region to be included in the amplicon using the same primers as the target molecule, and thus enable separation of the spike-in molecule and the endogenous target via electrophoresis. One would have a reasonable expectation of success given that Holck teaches that any qualitative PCR approach can easily incorporate the fluorescently labeled primers without changing the primer sets themselves and requires no additional steps be added to the traditional qualitative PCR methodologies (pg 952, col 1 last paragraph into col 2 first paragraph).
The claims of ‘543 in view of Holck do not teach that combining the peak intensities of the nucleic acid and combining the spike-in peak intensities comprises averaging said peak intensities and then computing the ratio, that the nucleic acid sample of the subject is a DNA sample, that each target is a chromosome of a set of chromosomes, that combining the peak intensities of the nucleic acid for a particular chromosome target comprises combining peak intensities for different fragments of the particular chromosome target, or that determining the abundance of the target comprises determining the presence or absence of an aneuploidy based on the computed ratios for two or more chromosomes. However, all of these features, determined through co-amplification of target and spike-in molecules, are known in the art as taught by Landry.
Landry teaches a method wherein an endogenous DNA target (such as a region on chromosome 21) is co-amplified with target-associated spike-in molecules and a reference target (a chromosome other than chromosome 21) and reference target-associated spike-in molecules (which reads on the plurality of other chromosome-specific ratios; paragraph [0060]). Landry teaches determining a ratio between each target/target associated pair intensity and each reference target/reference target-associated pair intensity (paragraph [0060]). Landry then teaches computing a ratio between the target-associated ratio and the reference-associated ratio, and if the target-associated ratio (first chromosome) relative to the reference-associated ratio (second chromosome) exceeds a “statistically significant threshold amount”, diagnosing aneuploidy (paragraph [0060]). Landry teaches averaging individual count ratios to determine abundance metrics (peak intensities; paragraph [0081, 00104-00105] and figure 3). Landry teaches that the target is a chromosome and the primers for amplification are chromosome-specific. Landry teaches combining peak intensities for different fragments of particular chromosome targets (paragraph [0060], Figure 3). Landry teaches that the sample is cell free DNA (paragraph [0032]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of ‘543 in view of Holck to apply said methodology to chromosome-specific targets as taught by Landry. One would be motivated to do so given the teachings by Landry that by determining the abundance metrics of specific chromosomes and comparing abundances between at least two chromosomes, one can diagnose diseases such as trisomy 21 (paragraph [0032]). One would be motivated to average peak intensities for targets and target-associated molecules given the teaching by Landry that averaging “can be used in determining an overall abundance metric with increased accuracy” (paragraph [0081]). One would be motivated to use different loci on the same chromosome and then combine said loci in determining the overall abundance metric for the target chromosome for the same reasoning (paragraph [0081]). One would have a reasonable expectation of success given that Landry is employing a similar methodology as that taught by ‘543 in view of Holck.
The claims of ‘543 in view of Holck and Landry do not teach co-amplifying chromosome-specific targets and spike-in molecules with chromosome-specific primers that have tails to generate amplicons of discrete lengths. However, use of tailed primers for generating amplicons of a discrete length of a set of discrete lengths is known in the art, as taught by Nygren.
Nygren teaches a method of determining the relative amount of a nucleic acid species in a sample (Abstract). Nygren teaches using “competitive PCR” to co-amplify a target and target-associated molecules with the same primer pair, and then using capillary electrophoresis to separate the fragments and determine relative abundance through computation of a ratio between the two (paragraphs [0020, 0106, 0259] and Figure 1). Nygren teaches that “amplified competitors can optionally be further distinguishable from one another through the use of tailed amplification primers” (paragraph [0112]) and that in cases when multiple genomic targets are being amplified in the same reaction “the primers that amplify the genomic DNA target sequences and corresponding competitor oligonucleotides each contain a 5’ tail of the same overall length” (paragraph [0165]). Nygren teaches that “In a multiplex amplification assay, for example, where a plurality of genomic DNA target sequences are co-amplified with a plurality of corresponding competitor oligonucleotides, the genomic DNA target sequences can all be of the same length, and the competitor oligonucleotides can be of the same or different lengths, such that the amplicons generated (through the use of tailed primers of varying length, for example) are distinct for each region assayed. In such cases, each region would be represented by two amplicon lengths (i.e. one amplicon length for the genomic DNA target(s) for that region and another amplicon length for the corresponding competitor oligonucleotide(s))” (paragraph [0184]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of ‘543 in view of Holck and Landry, who teach co-amplification of chromosome targets and target-associated molecules using labeled primers, to include tailed primers for co-amplification to generate discrete fragment lengths for both target and target-associated molecules for a given region, as taught by Nygren. One would be motivated to do so given that Nygren teaches that amplifying fragments of the same length and then varying the length with tailed primers allows for the removal of “unnecessary amplification bias” (paragraph [0260]). One would have a reasonable expectation of success given that the methodology of ‘543 in view of Holck and Landry already employs fragment separation via capillary electrophoresis (thus the length of the amplicons is being used to generate peak data), and the incorporation of the tailed primers for greater control of final amplicon length independent of the length of the target genomic region provides an additional level of controllable multiplex ability.
Claim 56, 62, 69, 74: Landry teaches aggregating the peak intensity abundance ratios across loci to generate a chromosome-specific ratio (paragraph [0105]).
Claim 57 and 70: Landry teaches that individual count ratios across different loci of a chromosome can be aggregated, then the spike-in individual count ratios can be aggregated, and then an overall abundance metric can be calculated from these aggregated intensities (paragraph [0105]).
Claims 58-59, 64-65, 71-72 and 76-77: Landry teaches that the sequence of dissimilarity in the spike-in molecule can be an insertion (such as a 5-base pair insertion) or a deletion (such as a one base pair deletion; paragraph [0093]).
Claim 60, 66, 73, and 78: Holck teaches that the synthetic competitor molecules have variation regions in the center of the molecules (Figure 1).
Claim 61: Holck teaches that each of the fluorescently labeled primers is associated with a color channel (Figure 2 and Table 3).
Claim 63 and 75: Landry teaches determining a ratio between each target/target associated pair intensity and each reference target/reference target-associated pair intensity (paragraph [0060]). Landry then teaches computing a ratio between the target-associated ratio and the reference-associated ratio, and if the target-associated ratio (first chromosome) relative to the reference-associated ratio (second chromosome) exceeds a “statistically significant threshold amount”, diagnosing aneuploidy (paragraph [0060]). Landry teaches averaging individual count ratios to determine abundance metrics (peak intensities; paragraph [0081, 0104-0105] and figure 3).
Claim 79 and 80: Landry teaches averaging peak intensities from the different fragment of the particular chromosome target, which necessarily includes aggregating the peak intensities first (paragraph [0081, 0104-0105].
US 12,183,437 B2
Claims 53 and 56-80 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-6, 9, 15, 17 and 18 of U.S. Patent No. US 12,183,437 B2 in view of Holck (Holck et al., European Food Research and Technology 2011; cited on PTO-892 of 2/6/2026), Landry (Landry et al., US 2019/0147980 A1; cited on IDS of 8/2/2023), and Nygren (US 2012/0264618 A1; cited on PTO-892 of 2/6/2026) according to the citations and rationales provided above.
US 11,646,100 B2
Claims 53 and 56-80 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 8-9, and 12 of U.S. Patent No. US 11,646,100 B2 in view of Holck (Holck et al., European Food Research and Technology 2011; cited on PTO-892 of 2/6/2026), Landry (Landry et al., US 2019/0147980 A1; cited on IDS of 8/2/2023), and Nygren (US 2012/0264618 A1; cited on PTO-892 of 2/6/2026) according to citations and rationales provided above.
Response to Remarks
Applicant has traversed the obviousness-type double patenting rejections as detailed in the Office Action of 2/6/2026 given the new claim limitations added in the most recent amendments. The previous double patenting rejections have been withdrawn and new double patenting rejections addressing said amendments have been presented above.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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