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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 2/11/2026 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 49-51 have been added. Claims 8 and 48 have been canceled. Claims 11-13, 19, 21-23, 30-31, 34-35, and 42 remain withdrawn. Claims 1-2, 5-7, 47, and 49-51 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/11/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
35 USC 112(b) Rejections
Claims 6 and 47-48 were rejected for various indefiniteness issues. Claim 48 has been canceled, and so this rejection has been rendered moot. In light of Applicant’s amendments to the claims submitted 2/11/2026, the rejections for claims 6 and 47 have been withdrawn.
35 USC 102 Rejections
Claims 1-2, 5-8, and 47-48 were rejected under 35 U.S.C. 102(a)(1) as being anticipated by Babiarz et al. (US 2016/0244838 A1), as evidenced by Qiagen (“QIAquick PCR Purification Kit for PCR Cleanup”).
In light of Applicant’s amendments to the claims submitted 2/11/2026, these rejections have been withdrawn for all currently pending claims. Claims 8 and 48 have been canceled, and so these claims have been rendered moot.
However, see new prior art rejections in the new grounds of rejections below.
Double Patenting Rejections
Claims 1-2, 6-8, and 47 were provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 2, 4-5, and 7-9 of copending Application No. 19/029,045 (reference application) in view of Babiarz (US 2016/0244838 A1), as evidenced by Qiagen (“QIAquick PCR Purification Kit for PCR Cleanup”).
In light of Applicant’s amendments to the claims submitted 2/11/2026, these rejections have been withdrawn for all currently pending claims, as the current claims are considered patentably distinct from those of the ‘045 application. Claim 8 has been canceled, so this rejection has been rendered moot.
Response to Applicant’s Arguments
Regarding the 35 USC 102 Rejections presented in the Final Rejection, Applicant argues that Babiarz, the primary reference used, does not teach the size selecting requirement of instant claim 1. Specifically, Applicant argues that the reference only teaches a purification of PCR products after targeted multiplex PCR, and not before, as presently claimed (Remarks, page 9, para. 1). Moreover, Applicant argues that the reference does not teach size-selection on amplified, adaptor-ligated DNA specifically (Remarks, page 9, para. 2). The reference, as evidenced by Qiagen, also does not allegedly teach size selection of 100-270 bp as claimed (Remarks, page 10, para. 1).
In light of Applicant’s amendments to the claims and further search and consideration of the prior art, the rejections involving the use of Babiarz as a primary reference have been withdrawn. Though Babiarz is used in the rejection of several claims below, the teachings of this reference utilized do not concern size selection. Therefore, Applicant’s arguments have been rendered moot.
Claim Interpretation
Regarding the specific size selection range discussed in instant claim 1, it is noted that the instant specification does not state that this range is critical or produces unexpected results. Para. 88 of the instant specification states that the 100-270 bp range is included in “one illustrative example,” and para. 200 describes size selection of 100-237 bp. Thus, in finding prior art that reads on this limitation, the guidance provided in MPEP 2144.05 will be used, where prior art that details ranges similar to or overlapping with the claimed size selection range will be considered to render the claimed range obvious.
Additionally regarding claim 1, step (c) states “obtaining a plurality of DNA fragments comprising a plurality of target loci each encompassing at least one of a plurality of individual-specific somatic mutations.” Due to the repeated use of the word “comprising,” this phrase will be interpreted as the plurality of DNA fragments also encompassing additional loci that do not contain individual-specific somatic mutations.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 5, 7, and 49 are rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017) as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 20191).
Alcaide teaches methods for detection of rare alleles in circulating tumor DNA (Abstract). This was done through the analysis of cell-free DNA (cfDNA) libraries (page 2, para. 2). Their general method is shown in Figure 1. First, tumor and/or liquid biopsies are used to identify somatic mutations for a particular sample. Then, utilizing a liquid sample, cfDNA is extracted, end-repaired, and ligated to adapters on each end. cfDNA is then amplified and subjected to two rounds of hybrid capture with biotinylated baits that are developed based on the specific somatic mutations in the samples. After capture, NGS sequencing is performed to analyze sequence reads to determine if the somatic mutants identified in the first tumor/liquid sample are present (see Figure 1 caption). Pages 14-15 note that the hybrid capture of multiple sequences occurred in single reaction mixtures (“Targeted enrichment and sequencing of cfDNA libraries.”).
In their specific methods, Alcaide states that after the ligation reaction, magnetic beads were used to clean the reaction mixture to remove adapter dimers (page 14, para. 1). These same magnetic beads were then used again after PCR was performed (see the same para., “Libraries were then purified using 0.8× volumes (48 µl) of Agencourt AMPure XP beads ensuring again that beads were fully resuspended in 75% ethanol during the washes.”). The PCR primers used by Alcaide are 42-69 nt long (page 14, para. 1). The adapters used by Alcaide are 48-50 nt long (page 13, para. 4). On page 11, para. 4 notes that the average size of cfDNA fragments is around 170 bp. Thus, adapter-ligated cfDNA is, on average, a little over 270 bp long (due to end-repair and A-tailing, see page 13, para. 6 and the Figure 1 caption). It is noted in Figure 1 that the PCR primers and the adapters do overlap in sequence near the end of the primer index sequence, meaning the portion of the primers that would be added to the adapter-ligated cfDNA during amplification is around 65 nt total (around 30 nt for the first primer and around 35 nt for the second based on the primer sequences on page 14, para. 1). Thus, adapter-ligated and amplified cfDNA is on average around 335 bp long. The magnetic beads used in Alcaide are AMPure XP magnetic beads (page 14, para. 1). Beckman Coulter notes that these beads are paramagnetic beads that can specifically remove primer dimers, and provide recovery of sequences that are over 100 bp long (see the information under “Cleanup and Size Selection”; instant claim 5). Alcaide’s use of the paramagnetic beads after PCR (in order to remove unused primer and potential primer dimers) would thus involve size selection of nucleotide sequences of greater than 100 bp to around 335 bp, which overlaps with the claimed range of 100-270 bp.
MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” As noted above in the “Claim Interpretation” section, as Applicant has not pointed out that their claimed range is critical or unexpected, the range for size selection described by Alcaide as evidenced by Beckman Coulter is considered to render obvious the claimed size selection range.
Thus, claims 1, 5, and 7 are prima facie obvious over Alcaide as evidenced by Beckman Coulter.
Regarding claim 2, Alcaide states that the samples initially gathered from patients were blood samples, where cfDNA was later isolated from plasma (page 13, para. 2).
Regarding claim 49, Alcaide specifically notes that their methods are designed to target mutations comprising SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2).
Claims 6, 47, and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017), as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 2019), and further in view of Babiarz et al. (US 2016/0244838 A1).
Alcaide as evidenced by Beckman Coulter teaches the methods of claims 1-2, 5, 7, and 49, as described above.
Regarding claims 6 and 47, Alcaide does not teach multiplex amplification after size selection, and instead utilizes hybrid capture, as described above. Alcaide also does not explicitly state measuring a large number of loci (e.g. Figure 1 shows 4 loci).
Babiarz teaches analyzing cfDNA from liquid samples (Abstract). These methods include analyzing circulating tumor nucleic acids via multiplex amplification (para. 56), and the reference includes multiple examples involving multiplex amplification of thousands of targets (e.g. paras. 109, 175, 188-190). Babiarz also alludes to matching mutations obtained from tumor samples with mutations in plasma samples utilizing multiplex amplification methods (paras. 119-120). In Example 7 of their methods, multiplex PCR is used to analyze chromosomal aneuploidy and copy number variation (para. 178). Blood samples were taken from cancer patients and cfDNA was isolated (paras. 181-182). Libraries were then generated by ligating adapters to DNA fragments and amplifying them (para. 187). Then, additional multiplex PCR was used for 3,168 SNPs to be examined in a single reaction (para. 188). This PCR is noted to be different than the initial amplification following adapter ligation (para. 187 notes PCR conditions involving 15 cycles, while paras. 189-190 note separate multiplex cycling conditions). Multiple amplification cycles occurred with primer pairs for each SNP (para. 189). The amplification products were sequenced via the Illumina HiSeq 2500 (paras. 189-191). Babiarz teaches that 32 CNVs were detected in their plasma samples from cancer patients (para. 210), where each CNV corresponds to multiple SNPs, where some regions contained over 100 SNPs (para. 193).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art that the multiplex amplification of Babiarz could be used in place of the hybrid capture of Alcaide as evidenced by Beckman Coulter. Babiarz teaches a similar method for manipulating cfDNA and targeting particular mutations as in Alcaide, including cfDNA isolation from a liquid sample, and the use of adapters, amplification, and sequencing. Alcaide details the process for the generation of biotinylated baits, but this also requires the use of PCR (and therefore, additional primers; page 14, para. 2). Therefore, rather than use the biotinylated baits that later require incubation with streptavidin and rely on PCR, it would be obvious that PCR could simply be used on the target cfDNA sequences, cutting down on the methodology while utilizing equipment already involved in the method. Such a method could be used with Illumina sequencing, as noted in para. 191 of Babiarz, which corresponds with the sequencing used in Alcaide, providing a reasonable expectation of success. This would essentially substitute the hybrid capture of Alcaide as evidenced by Beckman Coulter with multiplex amplification. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Both hybrid capture and multiplex amplification are known in the art, as evidenced by the references, and the substitution would have predictable results, as there would be particular SNPs that would be amplified and prepared for sequencing.
Thus, claim 6 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Additionally regarding claim 47, Babiarz shows that many different SNPs can be targeted at once. Alcaide teaches the use of patient-specific mutations (Figure 1), including SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2), and the number of potential patient-specific mutations possible to examine is only limited by the number of somatic mutations present in a patient. Babiarz notes that a large of number of SNPs can be present in a particular sample, and so it would be obvious to target additional SNPs in Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz, as creating a full profile of patient-specific mutations allows for enhanced mutation tracking overtime, which has implications for further diagnostics, prognoses, and treatment plans. For example, if the number of SNVs in a particular gene changes as cancer progresses in a patient, then practitioners can be aware of this trend, and monitor this gene closely in other patients with that same cancer. As Babiarz teaches that particular regions of interest have more than 100 SNPs, and teaches the analysis of over 3,000 SNPs in the working example described above, it would be prima facie obvious to specifically detect at least 100 patient-specific SNPs in the method of Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz. There would be a reasonable expectation of success as this would not involve changing the methodology steps or principles of Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz, but would simply involve focusing on additional targets.
Thus, claim 47 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Regarding claim 51, as noted above, Alcaide teaches measurement of target loci, though the reference does not explicitly state measuring a large number of loci (e.g. Figure 1 shows 4 loci).
Alcaide teaches the use of patient-specific mutations (Figure 1), including SNVs (Abstract, Table 3 caption, page 9, para. 5, and page 14, para. 2), and the number of potential patient-specific mutations possible to examine is only limited by the number of somatic mutations present in a patient. As noted above, Babiarz teaches measuring large numbers of SNPs in cfDNA and notes the utility of examining matching mutations in tumors and liquid samples for patients, and so with this guidance it would be obvious to target additional SNPs in Alcaide as evidenced by Beckman Coulter, as creating a full profile of patient-specific mutations allows for enhanced mutation tracking overtime, which has implications for further diagnostics, prognoses, and treatment plans. For example, if the number of SNVs in a particular gene changes as cancer progresses in a patient, then practitioners can be aware of this trend, and monitor this gene closely in other patients with that same cancer. As Babiarz teaches that particular regions of interest have more than 100 SNPs, and teaches the analysis of over 3,000 SNPs in their working example described above, it would be prima facie obvious to specifically detect at least 100 patient-specific SNPs in the method of Alcaide as evidenced by Beckman Coulter. There would be a reasonable expectation of success as this would not involve changing the methodology steps or principles of Alcaide as evidenced by Beckman Coulter but would simply involve focusing on additional targets.
Thus, claim 51 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Babiarz.
Claim 50 is rejected under 35 U.S.C. 103 as being unpatentable over Alcaide et al. (Scientific Reports, 2017), as evidenced by Beckman Coulter (“AMPure XP for PCR Purification”, 2019), and further in view of Dinakaran et al. (PLoS ONE, 2014) and Invitrogen (E-Gel SizeSelect II Agarose Gel, 2017).
Regarding claim 50, Alcaide as evidenced by Beckman Coulter teaches the methods of claims 1-2, 5, 7, and 49, as described above. This combination of references teaches size selection with paramagnetic beads, and not gel electrophoresis.
Dinakaran teaches measuring metagenomic profiling in cardiovascular disease patients (Abstract). Specifically, they focus on circulating cell-free DNA in patients (page 1, column 2, para. 1). Plasma DNA was extracted, amplified, and underwent barcoded shotgun sequencing (pages 2-3, “Extraction of plasma DNA,” “Whole genome amplification of circDNA,” and “Bar-coded shotgun sequencing of circDNA”). On page 3, the reference states that amplified DNA was fragmented to a size range of 100-600 bp with a maximum concentration around the 200 bp region. DNA fragments were then barcoded and adapters were ligated, and then additional size selection was performed with a 2% E-gel (“Bar-coded shotgun sequencing of circDNA”). Invitrogen notes that their E-Gel is a “fast and convenient method for DNA fragment library size selection as part of NGS library preparation workflows” (page 1, “Product description”). Page 4 notes the DNA ladder used with the gel goes from 1500-50 bp, and page 5 notes that library sizes with 100-400 base reads and peaks of 200-480 bp can be created.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to substitute the size selection and sequencing method described in Alcaide as evidenced by Beckman Coulter with that of Dinakaran and Invitrogen. Specifically, Alcaide as evidenced by Beckman Coulter teaches size selection with paramagnetic beads and Illumina sequencing, while Dinakaran and Invitrogen detail size selection with gel electrophoresis and Ion Torrent Sequencing (see page 3 of Dinakaran, column 2, para. 1 and Invitrogen page 5). Both types of size selection and sequencing are compatible with amplification and the use of adapters/barcodes, as is shown in both references. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Both methods of size selection and sequencing are known in the art, as shown in the references, and utilizing size selection with gel electrophoresis with Ion Torrent sequencing still allows for the processing of sequencing reads (see page 3, column 2, “Processing of Sequence reads” in Dinakaran). Thus, this substitution would provide predictable results (and this substitution would also have a reasonable expectation of success). Specifically, this substitution would result in the same general steps of Alcaide as evidenced by Beckman Coulter being used (cfDNA extraction, adapter ligation, PCR with sequences corresponding to adapters required for Ion Torrent sequencing, hybrid capture, size selection with gel electrophoresis to remove short sequences, and Ion Torrent sequences). As Invitrogen teaches that the E-gel can detect sequences below 100 bp (e.g. a user can see sequences at or below 100 bp via the DNA ladder), size selection can still be used to eliminate unused primers and primer dimers, and so size selection in a similar range to that described above in the rejection of claim 1 (i.e. a size range overlapping with the claimed size range) would be performed. Invitrogen also notes that their methods are fast and convenient, further motivating the ordinary artisan.
Thus, claim 50 is prima facie obvious over Alcaide, as evidenced by Beckman Coulter, and further in view of Dinakaran and Invitrogen.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
1 The attached webpage for this reference is from August 17, 2019. However, the reference itself notes that these beads were used in 2017 (see para. 2 under “Don’t Lose Critical Data”), and their use in Alcaide also indicates these beads were available in 2017, before the effective filing date of the claimed invention.