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.
Continued Examination Under 37 CFR 1.114
2. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 December 22, 2025 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 Claims 3 and 18 remain withdrawn as being drawn to a non-elected species.
Response to Arguments
3. Applicant’s arguments filed on December 22, 2025 regarding the previously made rejection of claims 1, 2, 4-7, 10-12, 15-17, 19-22, 25-27, and 30 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 apply to the new grounds of rejection, in which a new reference (Pakstis et al. Human Genetics 2010; 127: 315-324) has been added to the previous combination of references. The arguments have been fully considered and are discussed below.
Argument:
Applicant first argues that Quake fails to teach or suggest 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 6-8). More specifically, Applicant argues that Quake clearly does not contemplate performing the disclosed methods without prior genotype information since the reference only positively describes genotyping of donor and recipient to obtain a ‘predetermined marker profile’ that is then used to distinguish donor-derived nucleic acids from those of the recipient (Remarks, page 7). Applicant points to paras. 3, 69-70, 76-77, 88-89, 99, 101, 118, and 127 to support this argument (Remarks, page 7). Applicant also argues that reliance on MPEP 2123 in the Office’s rebuttal in the last Office action is misplaced because that section of the MPEP “concerns evaluation of disclosed embodiments, preferred or non-preferred, not the creation of hypothetical alternatives that are not described in the reference” (Remarks, page 7; emphasis in the original).
Response:
In response, the rejection has been withdrawn because Quake fails to provide an enabling disclosure for practicing the disclosed methods without the use of a predetermined marker panel.
It is true that Quake uses the phrase “in some embodiments” (e.g., in paras. 49, 69, and 70) to describe use of a predetermined marker panel, thereby suggesting that this is not always required, but Quake does not contain any detail as to how the method would be conducted without using a predetermined marker panel. Since the ordinary artisan would not have considered performing the method of Quake without using a predetermined set of markers to be routine (i.e., a change that could be made without additional guidance from Quake), Quake’s disclosure does not constitute an enabling disclosure. The rejection has been modified to add a new reference to address this deficiency since the additional references cited previously (Shoemaker and Varley & Mitra) do not address the deficiency in Quake.
Argument:
Applicant also argues that Quake fails to teach or suggest multiplexed PCR amplification of 20-1,000 polymorphic loci in the same reaction volume followed by barcoding PCR and high-throughput sequencing (Remarks, page 8). Here, Applicant argues that Quake only describes PCR, microarrays, and sequencing as three alternative options for use in genotyping, detecting or identifying donor and/or recipient nucleic acids, and/or quantifying donor-specific nucleic acids post-transplantation. Applicant points to paras. 76, 78, and 89 and also the working examples in Quake to support this argument (Remarks, page 8).
Response:
In response, the examiner agrees that Quake does not disclose a method that combines these three elements. The rejection does not allege that Quake teaches this combination of steps, though, and relies on two of the secondary references (Shoemaker and Varley & Mitra) to remedy this deficiency. The rejection acknowledges on pages 6-7 of the last Office action that Quake is deficient as to the barcoding PCR step and also as to the requirement in the claims for the method to combine multiplex PCR and sequencing and then goes on to describe why the ordinary artisan would modify the method of Quake in view of Shoemaker and also Varley & Mitra to arrive at the claimed methods.
Argument:
Lastly, Applicant presents arguments directed to Shoemaker and Varley & Mitra, each of which was cited as a secondary reference (Remarks, pages 8-10). Here, Applicant first argues that the secondary references do not remedy the deficiencies in Quake regarding the requirement in the independent claims for performing the method without prior knowledge of transplant donor or recipient genotypes (Remarks, page 8). Applicant also argues that neither secondary reference is described as suitable for use with the cell-free DNA that must be amplified and sequenced in the claimed methods (Remarks, pages 9-10). As to Shoemaker, Applicant argues that the reference is deficient because it only describes analysis of cellular DNA and fails to teach or suggest multiplexed targeted PCR amplification of cell-free DNA (Remarks, page 9). Therefore, Applicant argues, the ordinary artisan would have lacked motivation and reasonable expectation of success in combining the references to arrive at the claimed methods (Remarks, page 9). As to Varley & Mitra, Applicant argues that reference fails to cure the deficiencies of Quake and Shoemaker (Remarks, page 10).
Response:
In response to Applicant’s first argument on page 8, a new reference has been added to the rejection to address the deficiency of Quake with respect to performing the method in the absence of prior knowledge of genotypes.
As to Applicant’s arguments concerning Shoemaker, it is first noted that there is nothing in reference to indicate that the ordinary artisan could not use the cited teachings concerning multiplex targeted PCR, combining multiplex PCR and sequencing, or barcoding in the method of Quake. In other words, although the reference does discuss cellular DNA rather than cell-free DNA, the teachings in the reference concerning the advantages of combining multiplex PCR and sequencing, each of which is discussed in Quake, would not appear to be limited to methods performed with cellular DNA. That is, the ordinary artisan would have recognized that combining multiplex PCR and sequencing, each taught separately in Quake, would offer the same benefit of allowing for efficient analysis of large numbers of polymorphic loci.
Lastly, Applicant’s argument regarding Varley & Mitra was unpersuasive because the combination of references currently used is not deficient.
Information Disclosure Statement
4. The Information Disclosure Statement filed on December 22, 2025 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 Objections
6. Claim 31 is objected to because “chromosome” in line 2 should be replaced with “chromosomes.”
Claim Rejections - 35 USC § 112
7. 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
8. 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.
9. 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; newly cited).
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
10. 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.
11. 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).
12. 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; newly cited) and in view of Babiarz et al. (US 2016/0369333 A1; IDS reference, newly applied).
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.
13. 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; newly cited) and in view of Pakstis et al. (Human Genetics 2010; 127: 315-324; newly cited).
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.
14. 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; newly cited).
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
15. No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Angela Bertagna whose telephone number is (571)272-8291. The examiner can normally be reached 8-5, M-F.
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/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1637