Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
DETAILED ACTION
Status of the Claims
Claims 1-21 are pending and the subject of this FINAL Office Action.
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/27/2025 has been entered.
Applicants do not add new subject matter to the claims. Claim 21 is added, but was very clearly taught in the prior art cited. Specifically, claim 21 merely clarifies that “next-generation sequencing is sequencing-by-synthesis.” This is the most common sequencing at the time of the invention; and was clearly taught in the cited prior art (e.g. SHOEMAKER, para. 0124).
Priority
The claims receive a priority date of 05/18/2011 because the priority document filed on that day (13/300,235) is the first priority document to provide written description support of multiplex sequencing library preparation using 2-round barcoding.
Claim Rejections - 35 USC § 103 - Maintained
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-8, 10-16 and 18-21 are rejected under 35 U.S.C. § 103 as being unpatentable over SHOEMAKER (US 2008/0090239), in view of CHUU (US 2015/0218631), in view of LO (US 2009/0029377), MAY (US 2014/0227691) and MAY2 (WO 2010/115154).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date to multiplex barcode cfDNA (CHUU) instead of fetal cellular DNA (SHOEMAKER) for aneuploidy analysis with a reasonable expectation of success because cfDNA was a well-known non-invasive option instead of fetal cellular DNA.
As to claims 1-5, 10-13 and 18-20, SHOEMAKER teaches determining aneuploidy, e.g. triploidy (paras. 0140 & 0147, for example) by isolating DNA from the multiple maternal blood samples, wherein the DNA comprises fetal and maternal DNA (paras. 0002-06, 0071-72 & 0144); performing multiplex PCR on the fetal DNA to amplify 100 to 20,000 single nucleotide polymorphism (SNP) target loci in one reaction mixture, wherein the multiplex PCR is performed with primers designed to reduce primer dimer formation, and further performing universal PCR to amplify products of the multiplex PCR to obtain a non-naturally occurring composition of amplified DNA, wherein a sample barcode and a sequencing-compatible adaptor are incorporated into the amplified DNA during the multiplex PCR or the universal PCR (interrogating up to 1 million SNPs in parallel in multiplex reactions using primers pairs for amplifying multiple highly polymorphic SNPs on chromosomes 13, 18, 21 and X using “PCR primers are chosen to be multiplexible with other pairs (fairly uniform melting temperature, absence of cross-priming on the human genome, and absence of primer-primer interaction based on sequence analysis). The primers are designed to generate amplicons 10-200, 20-180, 40-160, 60-140 or 70-100 bp in size to increase the performance of the multiplex PCR,” followed by another PCR to add bin/sample tags/barcodes; paras. 0143-49, 0159 & 0225-30 and Figs. 4-5); analyzing the non-naturally occurring composition of amplified DNA by performing next-generation sequencing on multiple pooled indexed/barcoded samples (paras. 0118, 0121-22, 0151 & 0228-29) on the amplified DNA to obtain quantitative measurements of different alleles at the SNPs, and determining a ploidy state of the target chromosome using the quantitative measurements of different alleles at the SNPs (paras. 0010, 0015, 0193ff, 0235-36).
As to claims 7 and 15, SHOEMAKER teaches determining a ploidy state of a target chromosome of the fetus using the measured amounts of at least two of the target loci, wherein one or more of the target SNP loci map to chromosomes X, Y, 13, 18, and/or 21 and the method further comprises determining a ploidy state for chromosomes X, Y, 13, 18, and/or 21 using the measured amounts of the target loci (paras. 0142, 0152 & 0230).
SHOEMAKER does not explicitly teach cfDNA, using a reference genome, or estimating or calculating fetal fraction.
However, CHUU suggests as much, and a skilled artisan would have been familiar with techniques to accomplish this. For example, SHOEMAKER makes clear that his methods (using e.g. fetal cells) are non-invasive, and provides motivation to use other familiar non-invasive methods (paras. 0002-06). To this end, CHUU teaches another familiar non-invasive method to detect fetal aneuploidy using sequencing: cfDNA (Abstract; Fig. 26; Example 4). In fact, CHUU teaches sample barcoding and using common Illumina adaptors (with universal primers) to allow multiple samples and optimize sequence size for sequence reads on Illumina platforms (paras. 0115-16); and SHOEMAKER discloses familiar primer design parameters just like here (para. 0147, for example). Stated simply, cfDNA was a familiar target for adding familiar universal-primer sequencing adaptors with barcodes using primers to detect fetal aneuploidy.
CHUU also states throughout that thousands of target loci can be amplified using hundreds of primers (paras. 0004, 0009, 0017, 0062, 0065, 0067, 0070). In fact, the multiplex sequencing library preparation of CHUU Figure 26 was regularly used for multiplexing thousands of targets at a time. To this end, MAY2 demonstrates the same multi-round multiplex sequencing library preparation technique of CHUU Figure 26 (paras. 0114-0120), then states “In various embodiments, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000 or at least 10000 different target-specific primer pairs bearing the same first nucleotide tag and second nucleotide tag would be combined with the up to 2 or up to 4 outer barcode primers to generate multiple amplification products” (para. 0120). MAY2 even suggests this can be used for fetal DNA, just like in CHUU (para. 0132). Even more, MAY (same assignee and common inventors as MAY2) discusses this same sequencing library preparation technique of MAY2 specifically for fetal DNA (Abstract, paras. 0090-0114 (using same “inner” and “outer” primers of MAY2). Finally, MAY2 demonstrates that avoiding primer-dimers in multiplex amplification was routine (Table 1). Thus, a skilled artisan would have been motivated, with a reasonable expectation of success, to multiplex 100 or more cfDNA targets for sequencing library preparation utilizing familiar library preparation techniques such as in CHUU, MAY and MAY2.
As to obtaining amounts of the target loci based on SNP allele ratios, without prior knowledge of parental genotypes, and determining the fraction of fetal cell-free DNA in the maternal blood sample using the amounts of the target loci, this, too, was routine. In fact, CHUU specifically states “Methods for determining fetal aneuploidy using random sequencing techniques are described, for example, in U.S. Patent Application Publication Nos. 20090029377 [LO] . . . which are herein incorporated by reference in their entireties.” (para. 0090; see also paras. 0089-91). LO teaches obtaining amounts of the target loci based on SNP allele ratios, without prior knowledge of parental genotypes, and determining the fraction of fetal cell-free DNA in the maternal blood sample using the amounts of the target loci (using fetal fractional amounts based only on SNP allele ratios/frequencies/proportion/imbalance; paras. 0008-09, 0069, 0088, 0094, Fig. 2). In other words, CHUU expressly points to LO as a known, routine method to determine fetal aneuploidy.
As to claims 6 and 14, CHUU teaches estimating the fetal fraction of the cell-free DNA in the biological sample using the quantitative measurements of different alleles at the polymorphic loci (paras. 0090, incorporating US 61/296358, which matured into US 12/958347, Methods for determining fraction of fetal nucleic acids in maternal samples).
As to claims 8 and 16, CHUU teaches using a disomic reference genome and counting reads (paras. 0040-41, 0110-11).
In sum, it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date to apply familiar methods of detecting aneuploidy using multiplex sequencing library preparation to the library preparation of CHUU as suggested by CHUU with a reasonable expectation of success.
Claims 9 and 17 are rejected under 35 U.S.C. § 103 as being unpatentable over SHOEMAKER (US 2008/0090239), in view of CHUU (US 2015/0218631), in view of LO (US 2009/0029377), MAY (US 2014/0227691) and MAY2 (WO 2010/115154), in further view of SPARKS (US 20130288252).
The prior art teaches the elements of claims 1, 5 and 13-14 as explained. The prior art cited does not explicitly teach z-scores.
However this was familiar in the sequencing art to normalize and determine aneuploidy state (SPARKS, paras. 0289-92).
Thus, it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date to apply familiar aneuploidy sequencing data manipulation such as z-score to the aneuploidy data of the cited prior art with a reasonable expectation of success.
Previous Response To Arguments 06/26/2025
The rejections are maintained because Applicants fail to overcome the presumption of enablement for prior art. All prior art is presumed enabled. See In re Antor, 689 F.3d 1282, 1287-94 (Fed. Cir. 2012) (All prior art presumed enabled; Applicants must provide evidence to overcome this presumption); In re Morsa, 713 F.3d 104, 109-11 (Fed. Cir. 2013) (same). In order to overcome the presumption of enablement, Applicants are advised to apply the following guidance. "In order to anticipate, a prior art disclosure must also be enabling, such that one of ordinary skill in the art could practice the invention without undue experimentation. The standard for enablement of a prior art reference for purposes of anticipation under section 102 differs from the enablement standard under 35 U.S.C. § 112." Novo Nordisk Pharms., Inc. v. Bio-Tech. Gen. Corp., 424 F.3d 1347, 1355 (Fed. Cir. 2005) (internal citations omitted). While section 112 states that the specification must enable one skilled in the art to 'use' the invention, "section 102 makes no such requirement as to an anticipatory disclosure . . . . Rather, anticipation only requires that those suggestions be enabled to one of skill in the art." "Whether a prior art reference is enabling is a question of law based upon [the] underlying factual findings." Id (internal citations omitted.) It has also been held that "proof of efficacy is not required for a prior art reference to be enabling for purposes of anticipation." Impax Labs. Inc. v. Aventis Pharms. Inc., 468 F.3d 1366, 1383 (Fed. Cir. 2006). "Rather, the proper issue is whether the . . . patent is enabling in the sense that it describes the claimed invention sufficiently to enable a person of ordinary skill in the art to carry out the invention." Id. at 1383.
Applicants’ argument seems to be that SHOEMAKER (and CHUU, MAY and MAY2) does not enable multiplexing 1,000-20,000 primer pairs. Specifically, Applicants argue that SHOEMAKER fails to disclose a working embodiment of more than 100 primer pairs (Reply, pg. 8). However, as previously explained by the Examiner, SHOEMAKER in facts states that thousands to millions of primer pairs can be used in a multiplex to amplify thousands to millions of SNPs (paras. 0143-49, 0159 & 0225-30 and Figs. 4-5). If Applicants do not believe this is enabled by SHOEMAKER (which teaches the same generic outline as the instant claims), then it is Applicants’ duty to prove it. See Amgen Inc. v. Hoechst Marion Roussel, Inc., 314 F.3d 1313, 1355 (Fed. Cir. 2003) (In “patent prosecution the examiner is entitled to reject application claims . . . by a prior art patent without conducting an inquiry into whether or not that patent is enabled.” It then becomes Appellant’s burden to “overcome that rejection by proving that the relevant disclosures of the prior art patent are not enabled.”) Applicants have not done this.
As to CHUU, MAY and MAY2, the purpose for citing these references was not to fit them into SHOEMAKER like a puzzle, rather to demonstrate that cfDNA, using a reference genome, or estimating or calculating fetal fraction were all familiar sequencing techniques regularly used with success, including in directed sequencing contexts. Thus, Applicants’ arguments that CHUU, MAY and MAY2 would render SHOEMAKER unsatisfactory for its intended purpose are without merit.
Finally, designing primer pairs in multiplexes to reduce primer dimers is inherent in ALL multiplex PCRs. This is so well-known that claiming it fails to add anything to the claims. See e.g. Shen et al, (MPprimer: a program for reliable multiplex PCR primer design, BMC Bioinformatics volume 11, Article number: 143 (2010), 3/18/2010) (“there are several additional criteria which must be taken into account when considering multiplex PCR assay: 1) lack of primer dimerization between all of the primers; 2) similarity of the Tms of each primer . . . .”)
The rejections are maintained.
Response in Advisory Action 10/02/2025
The request for reconsideration has been considered but does NOT place the application in condition for allowance because: First, Applicants fail to appreciate all that SHOEMAKER teaches. SHOEMAKER teaches assay options that include multiplex PCR to label/tag/barcode multiple cells’ nucleic acids, including SNPs and nested PCR step, then pooling the multiple cells’ labeled/tagged/barcoded nucleic acids (0009, 0014, 0016, 0129, 0142, 0147-48 & 0226ff). In other words, the pooling is performed to pool single cells’ labeled/tagged/barcoded nucleic acids to create a mixture of multiple-cell labeled/tagged/barcoded nucleic acids for sequencing. As explained in SHOEMAKER, this pooling includes thousands of polymorphism or SNP targets in each cell.
CHUU, MAY and MAY2 also teach multiplex PCRs with thousands of primers. Applicants ignore the teachings in these references that explicitly discuss multiplex PCR. Simply put: why do these references discuss multiplex PCR (multiple primer pairs) if they don’t envision multiplex PCR?
As to MAY2, again Applicants fail to appreciate all that this reference teaches. MAY2 explicitly describes multiple PCRs (0053, 0085, 0120, 0210), including “multiplex 4-primer PCR using inner and outer primers” (para. 0053). In fact, paragraph 0210 explicitly states that “[i]n certain embodiments, multiplex detection is carried out in individual amplification mixture, e.g., in individual reaction chambers of a microfluidic device, which can be used to further increase the number of samples and/or targets that can be analyzed in a single assay or to carry out comparative methods, such as comparative genomic hybridization (CGH). In various embodiments, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10000 or more amplification reactions are carried out in each individual reaction chamber.” Thus, although some embodiments utilize one primer pair per chamber, yet others use multiple primer pairs per chamber, including thousands.
In sum, based on the prior art as a whole, including explicit segments thereof, the claimed multiplex amplification technique is obvious.
Response to Arguments
The rejections are maintained for the reasons provided in the Advisory Action 10/02/2025. Applicants’ arguments have not changed. They still argue, based on each prior art reference individually, that the prior art fails to teach and enable 2,000-SNP loci amplifications. However, the Examiner reminds Applicants what their claims require. A first generic “multiplex PCR” “to amplify 1,000-20,000 polymorphic loci,” followed by generic “universal PCR.” One of the PCRs include a generic “sample barcode” and generic “sequencing-compatible adaptor.” Simply put, the claimed assay is so generic that it encompasses the conventional use of conventional primers using conventional PCR to achieve conventional multiplex PCR with conventional “barcode” and conventional “adaptor.” The prior art demonstrates that these elements are conventional (i.e. very familiar), and regularly used with success. Achieving a multiplex of 1,000 loci is merely the conventional application of conventional multiplexing techniques (e.g. reducing primer dimers, similar primer melting temperatures, etc.). In fact, this is precisely what Applicants have stated on the record over and over again when confronted with enablement rejections for large multiplexes. The specification even admits that conventional multiplex techniques are utilized (e.g. reducing primer-dimer likelihood). There is nothing in the claims or specification that is new as to multiplexing PCR, much less sequencing library preparation.
To this end, the prior art as a whole demonstrates the motivation and skill to multiplex, including 1,000 loci and above with cfDNA for sequencing. The prior art also demonstrates that a first PCR (e.g. targeted preamplification with universal/common-primer-tailed primers), followed by universal pooled amplification was a well-known technique for sequencing library preparation of cfDNA. And so were barcodes for sequencing. The prior art as a whole renders the generic claims obvious.
Applicants are welcome to provide evidence that the prior art was not enabled for multiplex PCR of 2,000 loci or more; but until this is provided, the Examiner maintains that the prior art is presumed enabled for such multiplexes based on the teachings therein, even if no working example is provided therein. The prior art repeatedly teaches embodiments of multiplexes of thousands. Just like Applicants disclosure of conventional multiplex techniques is presumed enabled, the prior art is also presumed enabled. Even more, the burden to show the prior art is not enabled is higher that required to show Applicants’ own disclosure is not enabled. In light of all the facts, the prior art remains enabled, and demonstrates that multiplexing as claimed is obvious.
Prior Art
The following prior art is also pertinent to aneuploidy detection using multiplexed sequencing: US 20130310260; US 20140242588; US 20150167069; US 20150087535; US 20130157870; US 20130237431.
Conclusion
No claims are allowed.
All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681