DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application is being examined under the pre-AIA first to invent provisions.
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 prior art rejection set forth below 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.
Status of the Application
2. Applicant's response filed on April 29, 2026 has been entered.
Claims 1-7, 10-12, 15-22, 25-27, 30, and 31 are pending. Claims 3 and 18 remain withdrawn as being drawn to a non-elected species.
The objection to claim 31 has been withdrawn as moot in view of Applicant’s amendment to the claim.
The new matter rejection and the prior art rejections made previously have been maintained. Applicant’s arguments regarding these rejections are discussed below.
Response to Arguments
3. Applicant’s arguments filed on April 29, 2026 have been fully considered.
Rejection of claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, 30, and 31 under pre-AIA 35 U.S.C. 112, first paragraph (new matter)
Argument:
Applicant argues that the rejection should be withdrawn because the original disclosure, considered as a whole, provides support for the subject matter of claims 1 and 16, when examined in view of the species election (Remarks, pages 6-7). More specifically, Applicant argues that “[C]ombining separately disclosed features does not constitute new matter where the combination would have been apparent to one of ordinary skill in the art from the disclosure as a whole” (Remarks, page 7). Here, Applicant also argues that the specification supports the generic method recited in independent claims 1 and 16, where the method is practiced without prior genotype knowledge (Remarks, page 7). And, Applicant additionally argues that the rejection apparently conflates written description support with the presence of a working example and notes that this is not required, particularly since the disclosed mathematical framework can operate independent of the type of mixed sample (i.e., independent of whether the sample is obtained from a pregnant woman, a transplant recipient sample, or some other genetically distinct source) (Remarks, page 7). Applicant further identifies particular teachings on pages 26, 62, 64, 78-86, 88, 95, 112-119, and 129-132 of the specification as providing support for the claimed subject matter, particularly when considered as a whole (Remarks, pages 7-9).
Response:
These arguments have been fully considered, but they were not persuasive. The examiner agrees that the original disclosure must be considered as a whole and that combining separately disclosed elements does not necessarily constitute new matter if the combination would have been apparent when the disclosure is considered as a whole. The examiner also agrees that a working example is not necessarily required to demonstrate written description support.
In this case, though, the disclosure, including the portions cited by Applicant, when considered as a whole, does not reasonably indicate that performing any embodiments other than fetal DNA analysis in the absence of prior genotype knowledge was contemplated. Samples from a transplant recipient are only mentioned twice in the originally filed specification, once on page 88 and once on page 95. This clearly indicates that transplant samples are contemplated as suitable for use in the disclosed methods comprising amplification, sequencing, and analysis of polymorphic loci, but there is nothing to indicate that analyzing these samples in the absence of prior genotype knowledge concerning the donor and recipient, as required when the claims are considered in view of the species election, was contemplated. Instead, as discussed in the rejection, this concept is only discussed in the context of samples containing maternal and fetal DNA. It is also not clear that the ordinary artisan would have recognized that the calculations discussed on pages 112-119 and cited in Applicant’s arguments as providing support would apply to transplant samples or how to use the calculations in the context of transplant samples, particularly since this portion of the disclosure contains nothing to indicate that such generalization was contemplated. Indeed, this portion of the disclosure begins by stating, “Disclosed herein is a method for determining the ploidy state of a fetus given sequence data” (page 112, lines 6-7) and goes on to describe methods of determining the likelihood of monosomy, disomy, simple trisomy, or composite trisomy (pages 113-114). The ordinary artisan would not have considered this disclosure concerning fetal ploidy state hypotheses to apply to transplant samples to be tested for the amount of transplant donor DNA present in a large background of transplant recipient DNA.
The additional portions of the original disclosure cited in Applicant’s response do not remedy this deficiency. Therefore, the claims, considered in view of the species election, are still considered to contain new matter.
Lastly, Applicant’s additional arguments regarding dependent claims 2, 5-7, 10-12, 15-17, 20-22, 25-27, 30, and 31 on page 9 of the Remarks have been considered, but they are moot since the rejection does not allege that additional subject matter in these claims constitutes new matter.
Since Applicant’s arguments were not persuasive, the rejection has been maintained.
Rejection of claims 1, 2, 4, 7, 10, 12, 15-17, 19, 22, 25, 27, 30, and 31 under pre-AIA 35 U.S.C. 102(b) as being anticipated by Grskovic as evidenced by Pakstis
Applicant argues that the rejection should be withdrawn and presents two arguments regarding the rejection (Remarks, pages 10-12).
Argument 1: Grskovic does not qualify as prior art
Applicant first argues that the reference does not qualify as prior art (Remarks, page 10).
Response:
This argument was not persuasive. As discussed above, Applicant’s arguments regarding the new matter rejection were unpersuasive. Therefore, the effective filing date of the claims remains July 10, 2023, and Grskovic qualifies as prior art.
Argument 2: Grskovic does not teach all of the elements of independent claims 1 and 16
Applicant also argues that Grskovic fails to meet the requirement in independent claims 1 and 16 for the method to be performed “without prior knowledge of genotypes of the first and second individuals” (Remarks, pages 10-12). More specifically, Applicant argues that the method of Grskovic “requires inference of recipient genotype information from the data and then excludes certain loci based on that inferred genotype status,” which is not encompassed by the “without prior knowledge of genotypes” language in claims 1 and 16 (Remarks at page 10 – page 11, first para. and page 12, first full para.).1
Response:
This argument was not persuasive because “without prior knowledge of genotypes” language in claims 1 and 16 does not exclude the steps described by Grskovic in the “Percent dd-cfDNA Calculation” section on page 893. In other words, using population frequencies in the manner discussed on page 11 of the Remarks not required. 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).
Argument 3: The rejection improperly relies on Pakstis to reject claim 31
Applicant also argues that the rejection improperly uses reasoning that is “inferential and dependent on combining Grskovic with Pakstis” (Remarks, pages 11-12). In other words, Applicant argues, “Pakstis is not merely explaining Grskovic’s disclosure; it is being used to supply the missing ‘chromosome expected to be disomic’ limitation through combinatorial inference,” which is improper in an anticipation rejection (Remarks, page 12).
Response:
This argument was not persuasive. Contrary to Applicant’s argument, Pakstis is being used to explain Grskovic’s disclosure. As discussed in the rejection, the “SNP Selection and Primer Design” section on page 892 of Grskovic teaches analyzing 85 of the 92 SNPs previously identified by Pakstis. Grskovic does not go on to list the tested 85 SNPs, but the cited Pakstis reference does disclose the 92 SNPs referenced by Grskovic. At this point, it is incumbent upon the examiner to consider whether, in light of the additional information in Pakstis concerning the identity of the 92 SNPs from which Grskovic selected 85 for testing, Grskovic necessarily (i.e., inherently) analyzed at least some loci on a chromosome(s) expected to be disomic as required by claim 31. This is not improper “combinatorial reasoning” as Applicant argues because it simply seeks to determine what is inherently present (i.e., what is clearly and unequivocally present) in Grskovic by asking the following question: Can the 85 SNPs of Grskovic be selected from the 92 SNPs of Pakstis without including at least some SNPs from a chromosome(s) expected to be disomic? As can be seen in Table 1 of Pakstis, the 92 SNPs from which Grskovic selected 85 for further study include SNPs from all 22 autosomes, the vast majority, if not all, of which would be expected to be disomic in the patient population of Grskovic. Therefore, the rejection is not based on improper reasoning. Instead, it merely uses Pakstis to demonstrate what is inherent in the teachings of Grskovic.
Since Applicant’s arguments were not persuasive, the rejection has been maintained.
Rejections of claims 5, 6, 20, and 21 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Grskovic in view of Babiarz
Applicant argues that the rejection should be withdrawn (Remarks, page 13). Here, Applicant first argues that the rejection should be withdrawn because neither Grskovic nor Babiarz qualifies as prior art. Applicant also argues that the claimed ranges for the amplicon size do not lie “close” to the range disclosed in Grskovic as stated in the rejection, and instead, “are materially different…reflecting a fundamentally different primer design strategy” (Remarks, page 13).
These arguments were not persuasive.
First, Grskovic and Babiarz do, in fact, qualify as prior art. As discussed above, Applicant’s arguments regarding the new matter rejection were unpersuasive. Therefore, the effective filing date of the claims remains July 10, 2023, and Grskovic and Babiarz qualify as prior art.
Second, it is not unreasonable to consider the claimed ranges of “less than about 100 bp” and “about 65-80 bp” to lie “close” to the range of 100-130 nucleotides disclosed in Grskovic. All three ranges describe relatively short amplicons, and the claimed range of “less than about 100 bp” recited in claims 5 and 20 also overlaps with Grskovic’s range since “less than about 100 bp” encompasses lengths slightly greater than 100 bp. And, even if the ranges of Grskovic are not “close” to the claimed range, the rejection notes that the secondary reference (Babiarz) provides a clear reason to further reduce the amplicon size when practicing the method of Grskovic. See para. 383 of Babiarz, where the reference teaches that short fragments “may result in more efficient measurements of the desired polymorphic loci by only requiring short sequence reads.”
Since Applicant’s arguments were not persuasive, the rejection has been maintained.
Rejections of claims 11 and 26 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Grskovic in view of Pakstis
Applicant argues that the rejection should be withdrawn because Grskovic does not qualify as prior art (Remarks, page 14).
This argument was not persuasive because Grskovic does, in fact, qualify as prior art. As discussed above, Applicant’s arguments regarding the new matter rejection were unpersuasive. Therefore, the effective filing date of the claims remains July 10, 2023, and Grskovic qualifies as prior art. The rejection has been maintained.
Rejection of claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, 30, and 31 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Quake in view of Shoemaker and also in view of Varley & Mitra and further in view of Pakstis
Applicant argues that the rejection should be withdrawn (Remarks, pages 14-18). Here, Applicant presents four arguments as to why the rejection should be withdrawn.
Argument 1: Quake fails to teach or suggest performing the method without prior genotype knowledge
On pages 14-15, Applicant argues that Quake fails to teach or suggest performing the method without prior genotype knowledge since the methods disclosed in the reference “are fundamentally premised on establishing a predetermined marker profile by genotyping the transplant donor and recipient prior to cfDNA analysis” (Remarks, page 14). Applicant also notes that the working examples of Quake use a predetermined marker profile and that “There is no embodiment in Quake that describes how to quantify donor-derived cfDNA without such prior genotyping” (Remarks, page 15).
Response:
In response, the rejection does not allege that Quake discloses performing the disclosed methods without prior genotype knowledge. Instead, Pakstis was cited to remedy this deficiency in Quake.
Argument 2: Pakstis does not cure the deficiencies of Quake
On pages 15-16, Applicant argues that the 92 SNPs in the IISNP panel of Pakstis were chosen for forensic identification purposes, which is fundamentally different from the transplant monitoring recited in the claims at least because transplant monitoring requires quantification of the minority contributor fraction in a mixed sample (Remarks, page 15). Applicant also argues that Pakstis does not teach that the disclosed IISNP panel can be used for transplant monitoring as recited in the claims (Remarks, pages 15-16). Here, Applicant argues that “At most, Pakstis identifies SNPs useful for distinguishing unrelated individuals in an identification setting” (Remarks, page 15). Therefore, Applicant argues, the rejection is the result of impermissible hindsight (Remarks, page 16).
Response:
These arguments have been fully considered, but they were not persuasive. Applicant is correct that Pakstis does not discuss transplant monitoring, but as noted in the rejection, the reference does teach that the disclosed SNP panel is capable of distinguishing any two individuals, including closely related individuals (abstract, p. 316, and pp. 320-323). This, in combination with the teachings of Quake concerning how to calculate a minority fraction from measured allele amounts obtained during multiplex PCR or sequencing, would have provided the ordinary artisan with a reasonable expectation of success in using the SNP panel of Pakstis for transplant monitoring. As to motivation, as discussed in the rejection, the ordinary artisan would have recognized that using a predetermined panel of SNPs (i.e., the panel of Pakstis) would make the method of Quake more efficient by eliminating the need for an initial step of obtaining genotype information for the transplant donor and recipient. Thus, the rejection is not the result of improper hindsight and relies only on the teachings of the references in combination with the general ability to reason and make inferences possessed by the ordinary artisan.
Argument 3: Shoemaker and Varley & Mitra do not provide a reasonable expectation of success for multiplex PCR of cell-free DNA
As to Shoemaker, Applicant argues that the reference fails to teach or suggest highly multiplexed amplification of cell-free DNA since the reference “is directed to ‘rare cell analysis’ using cellular DNA obtained from a ‘mixed sample of rare and non-rare cells’” (Remarks, page 16). Applicant also argues that Shoemaker fails to provide a reasonable expectation of success for amplifying cell-free DNA from a mixed sample as required by the claims (Remarks, page 16).
As to Varley & Mitra, Applicant argues that the reference is “directed to mutation discovery in cancer genes using intact human genomic DNA, not cell-free DNA” (Remarks, page 17). Applicant additionally argues that Varley & Mitra do not teach or suggest applying the disclosed methods to cell-free DNA analysis and that the rejection results from improper hindsight given the differences between the “complex Nested Patch PCR workflow” of Varley & Mitra and the “simple barcoding PCR recited in the claims” (Remarks, page 17).
Response:
These arguments have been fully considered, but they were not persuasive.
As to Shoemaker, Applicant is correct that the reference describes amplification of cellular DNA rather than cell-free DNA, but there is nothing in the reference to indicate that the teachings regarding highly multiplexed amplification could not be applied to cell-free DNA, particularly since Shoemaker teaches that the disclosed methods may be used to generate short amplicons (para. 147).
Applicant’s arguments regarding technical challenges associated with multiplexed amplification of cell-free DNA were also unpersuasive for two reasons. First, the ordinary artisan would have been reasonably aware of such challenges, particularly when in possession of the teachings of the prior art references cited by Quake concerning cell-free DNA and amplification (paras. 40, 43, 74-75, and 77), and would have been able to apply the teachings of Shoemaker appropriately (e.g., by designing the primers to produce short amplicons) without undue experimentation. Second, the claims are very broadly written with respect to the primers and amplification conditions, with only an amplicon size requirement recited in dependent claims 5, 6, 20, and 21. This indicates that specific design strategies or amplification conditions other than accounting for the fragmented nature of cell-free DNA are not required for successful practice of the methods.
As to Varley & Mitra, Applicant is correct that the reference does not disclose analysis of cell-free DNA, but the ordinary artisan would nevertheless have recognized that the barcoding step disclosed in Figure 2 of the reference was universally applicable. This is because the barcoding step in Varley & Mitra occurs during a PCR used to add both barcodes and a sequencing adapter for a conventional next-generation sequencing platform (Fig. 2). In other words, the ordinary artisan would have recognized that the benefit of a sample-specific barcode—ability to pool samples and sequence in a single run—would not be limited to the particular samples of Varley & Mitra or to samples prepared by the Nested Patch PCR method of Varley & Mitra and would broadly extend to any other group of samples to be sequenced using the commercially available 454 sequencing platform. This conclusion is supported by the teachings of Varley & Mitra in the last paragraph on page 1844.
Argument 4: The four-reference combination is indicative of impermissible hindsight
Applicant argues on pages 17-18 of the Remarks that the rejection is the result of impermissible hindsight. Here, Applicant also argues that the different references are “each drawn from a different technological context and each being alleged to remedy different deficiencies in the others” (Remarks, page 17).
Response:
In response to Applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In this case, as discussed in greater detail above, the rejection provides a specific rationale for combining the teachings of the references to arrive at the claimed methods and uses only the teachings of the references in combination with the ability of the ordinary artisan to reason and make inferences.
Further, to the extent that Applicant is arguing that the Shoemaker, Varley & Mitra, and Pakstis references are nonanalogous art, it has been held that a prior art reference must either be in the field of the inventor’s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). In this case, all of the cited references are in the field of nucleic acid amplification. Each reference also pertains to at least one aspect of the particular problem with which the inventor was concerned. More specifically, Shoemaker is pertinent to multiplex amplification and also combining amplification and sequencing. Varley & Mitra pertains to combining amplification and sequencing. Pakstis pertains to the use of SNPs to distinguish individuals from one another.
Lastly, to the extent that Applicant is arguing that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
Since Applicant’s arguments were not persuasive, the rejection has been maintained.
Information Disclosure Statement
4. The Information Disclosure Statement filed on July 7, 2026 has been considered.
Priority
5. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) and 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
In this case, as discussed below, all of the claims under examination contain new matter. As a result, none of the prior-filed applications provides adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph the claims under examination, and the effective filing date of claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, 30, and 31 is July 10, 2023 (i.e., the filing date of the instant application).
Claim Rejections - 35 USC § 112
6. The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, 30, and 31 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Independent claims 1 and 16 were previously amended to require the method to be performed “without prior knowledge of genotypes of the first and second individuals.” See the response filed on July 8, 2025.
In view of the species election, which limits the extracted cell-free DNA to cell-free DNA from a transplant recipient that comprises DNA from the transplant donor and transplant recipient (see claim 4), all of the claims under examination require performing the amplification and sequencing method set forth in independent claims 1 and 16 and using allele amounts measured at a plurality of polymorphic loci to determine the amount of DNA from a first individual (i.e., the transplant donor) in the sample obtained from the second individual (i.e., the transplant recipient). This determination is made without prior knowledge of genotypes of the transplant donor and recipient.
Applicant’s response of July 8, 2025 pointed to the originally filed claims for support (Remarks, page 6).
Upon further consideration, independent claims 1 and 16, in view of the species election, contain new matter. This is because the original disclosure fails to provide support for the particular combination of elements and steps required by these claims in view of the species election. And more specifically, the original disclosure fails to describe how to determine the amount of DNA from a first individual in a sample obtained from the second individual when the first and second individual are, respectively, a transplant donor and a transplant recipient, and wherein the determination is made without prior knowledge of genotypes of the transplant donor and recipient.
The original claims do not provide support for this subject matter because they do not present these elements in combination. Instead, they are presented as separate options (see, e.g., original claims 4 and 14 as well as claims 19 and 29). The specification also fails to provide support for the aforementioned subject matter because it only discusses determining the amount of cell-free DNA from a first individual in a sample obtained from a second individual without prior genotype information in the context of prenatal diagnosis and ploidy calling (i.e., when the first individual is a fetus and the second individual is the mother) (see, e.g., pages 112-119 and 129-132 of the originally filed specification). In other words, there is nothing in the specification that describes how to apply the analysis to samples obtained from transplant recipient, particularly since transplant samples are only mentioned twice in the specification (pages 88 and 95). Thus, claims 1 and 16 contain new matter.
Claims 2, 4-7, 10-12, 15, 17, 19-22, 25-27, 30, and 31 also contain new matter since they depend from claim 1 or claim 16 and do not correct the new matter issue in those claims.
Claim Rejections - 35 USC § 102
7. The following is a quotation of the appropriate paragraphs of pre-AIA 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
8. Claims 1, 2, 4, 7, 10, 12, 15-17, 19, 22, 25, 27, 30, and 31 are rejected under pre-AIA 35 U.S.C. 102(b) as being anticipated by Grskovic et al. (Journal of Molecular Diagnostics 2016; 18: 890-902) as evidenced by Pakstis et al. (Human Genetics 2010; 127: 315-324).
Regarding claims 1, 2, 4, 7, 10, 12, 16, 17, 19, 22, 25, and 27, Grskovic discloses a method containing the following steps (see, e.g., the Materials and Methods section on pp. 891-893; see also Fig. 1):
(i) extracting cell-free DNA (cfDNA) of mixed origin from a plasma sample obtained from a transplant recipient, wherein the extracted cfDNA contains DNA from the transplant recipient and the transplant donor (pp. 891-892, the “Plasma Samples” section; see also Fig. 1 and p. 895, col. 2);
(ii) performing 15 cycles of multiplexed targeted PCR on the extracted cfDNA to amplify 266 SNP loci in a single reaction volume (p. 892, the “SNP Selection and Primer Design” and “Targeted Amplification and Sequencing” sections; see also Fig. 1 and p. 895, col. 2);
(iii) performing a barcoding PCR to add a sequencing tag and a sample index to the amplified DNA (p. 892, col. 2, the “Targeted Amplification and Sequencing”);
(iv) sequencing the resulting barcoded DNA and measuring an amount of each allele at the amplified SNP loci (pp. 892-893, the “Targeted Amplification and Sequencing” and “Sequencing Data Analysis and SNP Allele Counting” sections; see also p. 895, col. 2 and Fig. 1); and
(v) determining the amount of DNA from the transplant donor in the sample obtained from the transplant recipient (p. 893, the “Percent dd-cfDNA Calculation” section; see also p. 895, col. 2 and Fig. 1).
Further regarding claims 1 and 16, Grskovic teaches that the method is performed without prior genotype information for the transplant donor and recipient (see, e.g., the “SNP Selection and Primer Design” section on p. 892, col. 2).
Regarding claims 15 and 30, Grskovic teaches that the method further comprises pooling a plurality of differently indexed samples, sequencing the pooled samples together, and measuring allele amounts in the resulting sequencing data (pp. 892-893, the “Targeted Amplification and Sequencing,” “Sequencing Data Analysis and SNP Allele Counting,” and “Percent dd-cfDNA Calculation” sections).
Regarding claim 31, the method of Grskovic includes analysis of SNPs on a chromosome expected to be disomic (see, e.g., Fig. 1, where SNPs on chromosome 1 are analyzed). See also the “SNP Selection and Primer Design” section on p. 892, col. 2, where Grskovic references analyzing 85 of 92 SNPs previously identified by Pakstis. As can be seen in Table 1 of Pakstis, it is not possible to select 85 of the 92 SNPs in Table 1 of Pakstis without including a SNP on a chromosome expected to be disomic.
Claim Rejections - 35 USC § 103
9. The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
10. This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
11. Claims 5, 6, 20, and 21 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Grskovic et al. (Journal of Molecular Diagnostics 2016; 18: 890-902) as evidenced by Pakstis et al. (Human Genetics 2010; 127: 315-324) and in view of Babiarz et al. (US 2016/0369333 A1).
As discussed above, Grskovic as evidenced by Pakstis anticipates the methods of claims 1, 2, 4, 7, 10, 12, 15-17, 19, 22, 25, 27, 30, and 31.
Regarding claims 5, 6, 20, and 21, Grskovic teaches that the multiplex targeted PCR is performed with a plurality of primer pairs (p. 892, the “SNP Selection and Primer Design” section). This portion of Grskovic notes that the primer pairs are designed to produce a “median amplicon length of 109 nucleotides (minimum, 100 nucleotides; maximum, 130 nucleotides).” These values for the amplicon lengths lie close to the claimed range of “less than about 100 bp” recited in claims 5 and 20 and also lie close to the range of “about 65-80 bp” recited in claims 6 and 21.
It would have been prima facie obvious for one of ordinary skill in the art at the time of the invention to practice the method of Grskovic using primers capable of producing small amplicons (e.g., the claimed amplicons of less than 100 bp or about 65-80 bp) in view of the teachings of Babiarz. First, since Babiarz taught that DNA in plasma samples is typically fragmented and, on average, less than 200 bp in length (para. 367), the ordinary artisan would have recognized that cell-free DNA isolated from the plasma samples of Grskovic would also be highly fragmented and should be amplified using primers capable of generating short amplicons. Second, Babiarz provides motivation to use primers capable of producing short amplicons comprising polymorphic loci by teaching that short fragments “may result in more efficient measurements of the desired polymorphic loci by only requiring short sequence reads” (para. 383). Babiarz also provides motivation to generate amplicons with a length within the claimed ranges by disclosing example short amplicon lengths of “less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp” (para. 383). As well, since the claimed ranges lie close to the ranges disclosed in Grskovic and no evidence of unexpected results has been presented, a prima facie case of obviousness exists per MPEP 2144.05 I. Lastly, Babiarz provides a reasonable expectation of success by describing how to design and use such primers in a multiplex targeted amplification reaction (see, e.g., pages 42-50). Thus, the methods of claims 5, 6, 20, and 21 are prima facie obvious.
12. Claims 11 and 26 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Grskovic et al. (Journal of Molecular Diagnostics 2016; 18: 890-902) as evidenced by Pakstis et al. (Human Genetics 2010; 127: 315-324) and in view of Pakstis et al. (Human Genetics 2010; 127: 315-324).
As discussed above, Grskovic as evidenced by Pakstis anticipates the methods of claims 1, 2, 4, 7, 10, 12, 15-17, 19, 22, 25, 27, 30, and 31.
Grskovic does not teach that the polymorphic loci include indel loci, but this would have been obvious in view of Pakstis. More specifically, Pakstis teaches a panel of “92 SNPs for individual identification (IISNPs) with extremely low probabilities of any two unrelated individuals from anywhere in the world having identical genotypes” (abstract; see also Table 1). Pakstis also teaches that additional markers may be included in the panel (p. 323). Therefore, the ordinary artisan would have recognized that additional loci, such as indel loci, could also be used so long as they meet the criteria for inclusion in the panel disclosed in Pakstis (see, e.g., the abstract and pp. 320-323). The ordinary artisan would have had a reasonable expectation of success since Pakstis identified characteristics of useful markers (abstract and pp. 320-323). Thus, the methods of claims 11 and 26 are prima facie obvious.
13. Claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, 30, and 31 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Quake et al. (WO 2011/057061 A1) in view of Shoemaker et al. (US 2008/0090239 A1) and also in view of Varley & Mitra (Genome Research 2008; 18: 1844-1850) and further in view of Pakstis et al. (Human Genetics 2010; 127: 315-324).
The instant claims are drawn to a method for determining the amount of DNA from a first individual in a biological sample obtained from a second individual. Applicant has previously elected “cell-free DNA comprising DNA from a transplant” as the type of sample for examination. Therefore, the first individual is a transplant donor, and the second individual is a transplant recipient. The methods comprise amplifying a plurality of polymorphic loci in cell-free DNA extracted from the biological sample by multiplexed PCR, performing a barcoding PCR, using high-throughput sequencing to sequence the resulting barcoded amplification products, measuring the amount of each allele at the polymorphic loci, and determining the amount of DNA from the first individual in the biological sample. Independent claims 1 and 16 additionally require the method to be performed “without prior knowledge of genotypes of the first and second individuals.”
Quake discloses methods for diagnosing or predicting transplant rejection in a transplant recipient based on the amount of donor nucleic acid present in a sample obtained from the transplant recipient (see, e.g., the abstract and paras. 3, 5, 6, 8-10, 42, and 43).
Regarding independent claims 1, 4, 16, and 19, the method of Quake comprises the following steps:
(a) extracting cell-free DNA of mixed origin from a biological sample obtained from a transplant recipient, wherein the cell-free DNA comprises DNA from the transplant donor and the transplant recipient (see, e.g., paras. 3, 5, 8-10, and 121); and
(b) measuring the amount of alleles at each of a plurality of polymorphic loci in the cell-free DNA to determine the amount of DNA from the donor in the sample obtained from the transplant recipient (see, e.g., paras. 3, 5, 8-10, 46-49, 60-63, 98-107, and 110).
Further regarding claims 1 and 16, Quake teaches that the method may include analysis of large numbers of polymorphic loci (see, e.g., paras. 61-63). The ranges for the number of polymorphisms to be analyzed disclosed in these portions of Quake overlap with or encompass the ranges recited in the instant claims 1 and 16. Quake also teaches that the polymorphic loci may be analyzed by PCR (e.g., multiplex PCR) or sequencing (see, e.g., paras. 76 and 78, respectively; see also para. 105). As well, the sequencing may be high-throughput sequencing (see, e.g., paras. 78-80).
Regarding claims 2 and 17, Quake teaches that the sample may be a blood, serum, plasma, or urine sample (see, e.g., paras. 43, 48, and 104).
Regarding claims 10, 11, 25, and 26, Quake teaches that the polymorphic loci may comprise SNP loci or indel loci (see, e.g., para. 61).
Quake does not teach all of the elements of the rejected claims. First, Quake does not clearly combine multiplex PCR and sequencing as required by independent claims 1 and 16. Second, Quake does not disclose barcoding as also required by independent claims 1 and 16. Third, although Quake discloses analysis of a large number of polymorphic loci, the reference is silent as to the number of loci amplified by multiplex PCR. Claims 1 and 16 require using multiplex targeted PCR to amplify 20-1000 or 50-500 polymorphic loci, respectively. Lastly, Quake only provides an enabling disclosure for using a predetermined panel of polymorphic loci to determine the amount of donor DNA in the sample obtained from the transplant recipient.
Like Quake, Shoemaker also discloses methods for non-invasive detection of nucleic acids of interest in samples of mixed origin. The methods disclosed by Shoemaker include amplification, which may be multiplexed PCR, followed by high-throughput sequencing (see, e.g., paras. 14-15 and paras. 226-234). Quantitative analysis of large numbers of polymorphic markers (e.g., SNPs) is also disclosed (see, e.g., paras. 15 and 159).
Further regarding claims 1, 5, 6, 16, 20, and 21, Shoemaker discloses multiplex PCR amplification of as many as 100 SNPs (see, e.g., para. 147). The resulting amplicons may be 10-200, 20-180, 40-160, 60-140, or 70-100 bp in length (para. 147). These ranges overlap with claimed ranges of “less than about 100 bp” and “about 65-80 bp.”
Shoemaker does not disclose a separate barcoding PCR since the analogous “locater tag” is added during multiplex PCR (see, e.g., paras. 115-118), but Varley & Mitra disclose a method that comprises the following steps: (i) multiplex PCR; (ii) barcoding PCR; and (iii) next-generation sequencing of the barcoded PCR products (see, e.g., the abstract, Figs. 1-2, and p. 1845). As noted in Figure 2 of Varley & Mitra, the barcoding PCR adds a sample-specific barcode as well the sequencing tag recited in claims 12 and 27. Further regarding claims 15 and 30, Varley & Mitra teach that barcoding allows for pooling of a plurality of different samples and subsequently sequencing the pooled samples together since the barcode will allow sample identification (see, e.g., p. 1845).
Neither Shoemaker nor Varley & Mitra discuss determining the amount of DNA from one individual in a sample obtained from another individual in the absence of prior genotype knowledge, but Pakstis teaches a panel of “92 SNPs for individual identification (IISNPs) with extremely low probabilities of any two unrelated individuals from anywhere in the world having identical genotypes” (abstract). See also Table 1 for the list of IISNPs. Further regarding new claim 31, the SNPs in Table 1 of Pakstis include SNPs on chromosomes expected to be disomic.
It would have been prima facie obvious for one of ordinary skill in the art at the time of the invention to combine multiplex PCR and sequencing as described in Shoemaker when practicing the methods of Quake. As noted above, Quake discloses using multiplex PCR or high-throughput sequencing to measure the amounts of alleles from a transplant donor in a sample obtained from a transplant recipient, but fails to clearly teach combining these two methods. Shoemaker provides a rationale for doing so, though, since the teachings in that reference (e.g., in Example 4 at paras. 225-236) would have indicated to the ordinary artisan that multiplex PCR and high-throughput sequencing could be combined to provide a method for efficiently analyzing a large number of polymorphic loci of interest in a sample of mixed origin. The ordinary artisan would have had a reasonable expectation of success in view of the guidance provided by Shoemaker concerning multiplex PCR, the guidance provided by Shoemaker and Quake concerning high-throughput sequencing, and the general knowledge available to the ordinary artisan concerning these techniques.
Further regarding the number of loci analyzed by the method, as noted above, Quake discloses ranges for the number of loci that overlap with the ranges recited in claims 1 and 16, and no evidence of unexpected results has been presented with respect to the claimed numbers of loci. Similarly, Shoemaker discloses ranges for the size of multiplex PCR products that overlap with the length ranges recited in claims 5, 6, 20, and 21, and no evidence of unexpected results has been presented with respect to amplicon length. This is sufficient to establish a prima facie case of obviousness for the ranges recited in claims 1, 5, 6, 16, 20, and 21 per MPEP 2144.05 I.
It also would have been prima facie obvious to further include a barcoding PCR to add a sample index and a sequencing tag when practicing the method suggested by Quake in view of Shoemaker. As noted above, Shoemaker discloses barcoding, but not in a separate PCR. The teachings of Varely & Mitra, though, indicate that barcoding may be performed in a separate PCR that also adds a sequencing tag (see, e.g., Fig. 2 and p. 1845). The ordinary artisan would have recognized from these teachings in the art that a barcoding/sequencing tag addition step could be performed either during a multiplex PCR suggested by Quake in view of Shoemaker or in a separate step conducted between the multiplex PCR and the sequencing step, and accordingly, would have been motivated to select either method of barcode/sequencing tag addition with a reasonable expectation of success. The ordinary artisan would have been particularly motivated to include barcoding since each of Shoemaker and Varley & Mitra taught that sample barcoding allowed for pooling different samples and sequencing them together as recited in claims 15 and 30 (see, e.g., Shoemaker at para. 228; see Varley & Mitra at p. 1845).
Lastly, it would have been prima facie obvious to use the IISNPs panel disclosed in Pakstis when practicing the method suggested by Quake in view of Shoemaker and Varley & Mitra. The ordinary artisan would have been motivated to do so to eliminate the need to obtain genotype information for the transplant donor and recipient when practicing the method of Quake and would have had a reasonable expectation of success since Pakstis taught that the disclosed panel of SNPs could be used to distinguish any two individuals, including closely related individuals (see, e.g., the abstract and pages 316 and 320-323).
Thus, the methods of claims 1, 2, 4-6, 10, 12, 15-17, 19-21, 25, 27, 30, and 31 are prima facie obvious.
Further regarding claims 7 and 22, the cited references do not specify the number of PCR cycles used for the multiplex PCR step, but as noted in MPEP 2144.05 II, optimizing results-effective variables is prima facie obvious in the absence of unexpected results. In this case, the number of PCR cycles would have been recognized as a results-effective variable by the ordinary artisan, and no evidence of unexpected results has been presented with respect to the claimed range of 10-40 PCR cycles. Thus, this range is prima facie obvious.
Finally, further regarding claims 11 and 26, it also would have been prima facie obvious to include additional polymorphic loci, such as indel loci, in the panel disclosed in Pakstis. Pakstis provides motivation to do so by teaching that additional markers may be included in the panel (p. 323). The ordinary artisan would, therefore, have recognized that additional loci, such as indel loci, could also be used so long as they meet the criteria for inclusion in the panel disclosed in Pakstis (see, e.g., the abstract and pp. 320-323). The ordinary artisan would have had a reasonable expectation of success since Pakstis identified characteristics of useful markers (abstract and pp. 320-323). Thus, the methods of claims 11 and 26 are prima facie obvious.
Conclusion
14. No claims are allowable.
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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/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1637
1 It is noted that the portions of Grskovic quoted on page 11 of the Remarks appear on page 893 of the reference rather than page 894.
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