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 .
DETAILED ACTION
Applicant’s response filed on March 3, 2026 is acknowledged and has been entered. Claims 1-14 and 16-21 are pending. Claims 1, 11 and 21 are amended. Claim 15 is canceled.
Claims 1-14 and 16-21 are discussed in this Office action.
All of the amendments and arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons discussed below. Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
This action is made FINAL.
Prior Grounds of Rejection
Rejection updated to address amendment to the claims using Gnirke reference, in part (regarding the depth of read limitation).
Priority
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)
The disclosure of the prior-filed application, Application No. 61395850 (‘850 application), 61398159 (‘159 application), 61426208 (‘208 application) and 61462972 (‘972 application), 61448547 (‘547 application) and 61516996 (‘996 application) and 61571248 (‘248 application) fail to provide 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 for one or more claims of this application as amended. In the ‘850 application, while the disclosure teaches amplification of cell free nucleic acids via PCR, the tagging and detection of cancer or tumor associated mutations was not clearly supported. In the ‘159 and the ‘208 applications, while the applications provide support for amplification of cell free nucleic acids and including mutations and sequencing, the disclosures do not provide support for tagging, barcodes, enrichment, cancer or hybrid capture. While the ‘972 application provides support for cell free nucleic acids, multiplex amplification and sequencing, the application does not provide support for barcoding or enrichment. While the ‘547 application and ‘996 applications provides support for many aspects of the method, as claimed, the disclosures do not provide support for the amendment to the claims wherein molecular barcodes in the sequence reads are used to identify sequence reads from the same isolated cell-free DNA molecule. Therefore, the claims are entitled to an earliest priority date of October 3, 2011 as recited in the 61542508 application (‘508 application) and also the 10017812 patent.
Claim Rejections - 35 USC § 103
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.
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, 6-12 and 16-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pieprzyk et al. (US PgPub 20140186827; July 2014), McCloskey et al. (PgPub 20070020640; January 2007), and Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189).
With regard to claim 1, Pieprzyk teaches a method for enriching and sequencing cell-free DNA, comprising:
tagging each strand of cell-free DNA molecules isolated from a single biological sample with barcodes to obtain a plurality of barcoded DNA molecules comprising a plurality of target loci (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well-designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included);
amplifying the plurality of barcoded DNA molecules by universal amplification to obtain a sequencing library (paragraph 71, which points to Figure 11A-B and Example 13, paragraph 501, where universal tags are included; see also Example 13 and Example 14, where universal or common sequences are used in amplification; see also paragraph 173, where sequencing libraries are also generated in different embodiments);
enriching for the at least 50 target loci from the sequencing library (paragraph 173, where samples are enriched for amplifiable cell-free DNA); and
sequencing to sequence the enriched target loci and determine whether the target loci to obtain sequence reads of the at least 50 target loci and determine whether the target loci comprise a cancer-associated mutation (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included);
wherein the molecular barcodes in the sequence reads are used to identify sequence reads from the same isolated cell-free DNA molecule and to measure the relative amount of each allele of the target loci in the biological sample (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included; see paragraph 298 where “detecting the presence or amount of one or more target nucleic acids in a nucleic acid sample. Thus, for example, these methods are applicable to identifying the presence of particular polymorphisms (such as SNPs), alleles, or haplotypes, or chromosomal abnormalities”).
With regard to claim 2, Pieprzyk teaches a method of claim 1, wherein the single biological sample is a blood, plasma, serum, or urine sample (paragraph 2, 57, 172 where the sample includes blood, plasma or urine).
With regard to claim 8, Pieprzyk teaches a method of claim 1, wherein the cell-free DNA are tagged with the molecular barcodes through ligation (paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 9, Pieprzyk teaches a method of claim 1, wherein sequence reads originating from the same original molecule are identified using the molecular barcodes (paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 10, Pieprzyk teaches a method of claim 1, wherein the universal amplification introduces a sample-specific barcode, and wherein amplified DNAs of multiple samples are pooled together and sequenced in a single sequencing lane (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 11, Pieprzyk teaches a method for enriching and sequencing cell-free DNA, comprising:
tagging cell-free DNA molecules isolated from a single biological sample with a plurality of different molecular barcodes to obtain a plurality of barcoded DNA molecules comprising a plurality of target loci wherein cell-free DNA molecules comprising the same target loci are each tagged with barcodes, wherein the plurality of target loci comprises at least 50 target loci (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included);
wherein cell-free DNA molecules comprising the same target loci are each tagged with a different molecular barcode and can be distinguished from one another upon sequencing of the molecular barcodes, wherein the plurality of target loci comprises at least 50 target loci (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included);
amplifying the plurality of barcoded DNA molecules by universal amplification to obtain a sequencing library (paragraph 71, which points to Figure 11A-B and Example 13, paragraph 501, where universal tags are included; see also Example 13 and Example 14, where universal or common sequences are used in amplification; see also paragraph 173, where sequencing libraries are also generated in different embodiments);
enriching for the at least 50 target loci from the sequencing library using hybrid capture probes (paragraph 173, where samples are enriched for amplifiable cell-free DNA);
and performing sequencing to sequence the enriched target loci to obtain sequence reads of the at least 50 target loci and determine whether the target loci comprise a cancer-associated mutation, wherein the cell free DNA from a single biological sample are tagged with a plurality of different molecular barcodes wherein the molecular barcodes in the sequence reads are used to identify sequence reads from the same isolated cell-free DNA molecule and to measure the relative amount of each allele of the target loci in the biological sample (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included; see paragraph 298 where “detecting the presence or amount of one or more target nucleic acids in a nucleic acid sample. Thus, for example, these methods are applicable to identifying the presence of particular polymorphisms (such as SNPs), alleles, or haplotypes, or chromosomal abnormalities”).
With regard to claim 12, Pieprzyk teaches a method of claim 11, wherein the single biological sample is a blood, plasma, serum, or urine sample (paragraph 2, 57, 172 where the sample includes blood, plasma or urine).
With regard to claim 18, Pieprzyk teaches a method of claim 1, wherein the cell-free DNA are tagged with the molecular barcodes through ligation (paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 19, Pieprzyk teaches a method of claim 1, wherein sequence reads originating from the same original molecule are identified using the molecular barcodes (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 20, Pieprzyk teaches a method of claim 1, wherein the universal amplification introduces a sample-specific barcode, and wherein amplified DNAs of multiple samples are pooled together and sequenced in a single sequencing lane (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included).
With regard to claim 21, Pieprzyk teaches a method for enriching and sequencing cell-free DNA, comprising:
tagging cell-free DNA molecules isolated from a single biological sample with barcodes to obtain barcoded DNA comprising a plurality of target loci, wherein cell-free DNA molecules comprising the same target loci are each tagged with a different molecular barcode and can be distinguished from one another upon sequencing of the molecular barcodes;
amplifying the barcoded DNA by universal amplification to obtain a sequencing library;
enriching for the plurality of target loci from the sequencing library using target-specific primers or probes, wherein the target-specific primers or probes each comprises a nucleic acid sequence complementary to a specific target DNA sequence of the target loci; and
performing high-throughput sequencing to sequence the enriched target loci to obtain sequence reads of the at least 50 target loci with a depth of read of at least 200 per target locus and determine the presence of a cancer-associated mutation at the target loci, and wherein the molecular barcodes in the sequence reads are used to identify sequence reads from the same isolated cell-free DNA molecule and to determine a relative amount of each allele at the target loci in the biological sample, wherein the plurality of target loci comprises at least 50 target loci (see Example 10, where cell free nucleic acids are used for determination of fetal aneuploidy and which recites the reasons why barcodes are important to the implementation of the method of Pieprzyk as applies to the determination of fetal aneuploidy; specifically at Example 10, Pieprzyk teaches that the inclusion of unique barcodes allows for multiplexing, addresses “depth issues associated with unequal sequence coverage” that “are avoided with well designed loci”. As a final step, “reaction products are identified and counted to make the determination of aneuploidy”; see also paragraph 98; see also paragraph 285, where unique molecular barcodes are included; see paragraph 298 where “detecting the presence or amount of one or more target nucleic acids in a nucleic acid sample. Thus, for example, these methods are applicable to identifying the presence of particular polymorphisms (such as SNPs), alleles, or haplotypes, or chromosomal abnormalities”).
Regarding claim 1, 11 and 21, while Pieprzyk teaches the inclusion of barcodes, Pieprzyk does not teach the step wherein the extracted cell-free DNA from a single biological sample is tagged with a plurality of different molecular barcodes and can be distinguished from one another upon sequencing of the molecular barcodes.
With regard to claim 1, 11 and 21, McCloskey teaches wherein the extracted cell-free DNA from the biological sample is tagged with a plurality of different molecular barcodes (paragraph 21, where "random barcode" refers to an arbitrary sequence that can uniquely identify a target nucleic acid in an experiment, and whose sequence is unknown at the start of the experiment; later in the same paragraph, McCloskey notes “a second sequence of 7 random nucleotides N selected from A, G, C, and T will provide a maximum of 47 or 16,384 unique barcodes. In some embodiments, the length of the second sequence is between 3 and 30 nucleotides, such as between 5 and 25 nucleotides or between 7 and 13 nucleotides”; see Example 1, p 6, paragraph 53, where barcodes are useful in identifying unique sequences; see also Example 2, p 7, paragraph 63, where barcodes are again useful in identification of unique sequences; see also Table 1, for example).
With regard to claim 6, McCloskey teaches a method of claim 1, wherein the cell-free DNA are tagged with up to 1024 molecular barcodes (paragraph 21, where "random barcode" refers to an arbitrary sequence that can uniquely identify a target nucleic acid in an experiment, and whose sequence is unknown at the start of the experiment; later in the same paragraph, McCloskey notes “a second sequence of 7 random nucleotides N selected from A, G, C, and T will provide a maximum of 47 or 16,384 unique barcodes. In some embodiments, the length of the second sequence is between 3 and 30 nucleotides, such as between 5 and 25 nucleotides or between 7 and 13 nucleotides”; see Example 1, p 6, paragraph 53, where barcodes are useful in identifying unique sequences; see also Example 2, p 7, paragraph 63, where barcodes are again useful in identification of unique sequences; see also Table 1, for example).
With regard to claim 7, McCloskey teaches a method of claim 1, wherein the cell-free DNA are tagged with 1024- 65536 molecular barcodes (paragraph 21, where "random barcode" refers to an arbitrary sequence that can uniquely identify a target nucleic acid in an experiment, and whose sequence is unknown at the start of the experiment; later in the same paragraph, McCloskey notes “a second sequence of 7 random nucleotides N selected from A, G, C, and T will provide a maximum of 47 or 16,384 unique barcodes. In some embodiments, the length of the second sequence is between 3 and 30 nucleotides, such as between 5 and 25 nucleotides or between 7 and 13 nucleotides”; see Example 1, p 6, paragraph 53, where barcodes are useful in identifying unique sequences; see also Example 2, p 7, paragraph 63, where barcodes are again useful in identification of unique sequences; see also Table 1, for example).
With regard to claim 16, McCloskey teaches a method of claim 1, wherein the cell-free DNA are tagged with up to 1024 molecular barcodes (paragraph 21, where "random barcode" refers to an arbitrary sequence that can uniquely identify a target nucleic acid in an experiment, and whose sequence is unknown at the start of the experiment; later in the same paragraph, McCloskey notes “a second sequence of 7 random nucleotides N selected from A, G, C, and T will provide a maximum of 47 or 16,384 unique barcodes. In some embodiments, the length of the second sequence is between 3 and 30 nucleotides, such as between 5 and 25 nucleotides or between 7 and 13 nucleotides”; see Example 1, p 6, paragraph 53, where barcodes are useful in identifying unique sequences; see also Example 2, p 7, paragraph 63, where barcodes are again useful in identification of unique sequences; see also Table 1, for example).
With regard to claim 17, McCloskey teaches a method of claim 1, wherein the cell-free DNA are tagged with 1024- 65536 molecular barcodes (paragraph 21, where "random barcode" refers to an arbitrary sequence that can uniquely identify a target nucleic acid in an experiment, and whose sequence is unknown at the start of the experiment; later in the same paragraph, McCloskey notes “a second sequence of 7 random nucleotides N selected from A, G, C, and T will provide a maximum of 47 or 16,384 unique barcodes. In some embodiments, the length of the second sequence is between 3 and 30 nucleotides, such as between 5 and 25 nucleotides or between 7 and 13 nucleotides”; see Example 1, p 6, paragraph 53, where barcodes are useful in identifying unique sequences; see also Example 2, p 7, paragraph 63, where barcodes are again useful in identification of unique sequences; see also Table 1, for example).
Regarding claims 1 and 11, while Pieprzyk teaches analysis of mutations using sequencing, Pieprzyk also does not teach using hybrid capture probes. Further, while Pieprzyk teaches sequencing, Pieprzyk does not teach sequencing with a depth of read of at least 200 per target locus.
With regard to claim 1 and 11, Gnirke teaches sequencing with a depth of read of at least 200 per target locus (p 185, col. 1, where “The average depth of coverage for the 0.75 million genome bases covered by bait in the four target regions was 221”); and
enriching for a plurality of target loci using hybrid capture probes wherein the hybrid capture probes each comprises a nucleic acid sequence complementary to a specific target DNA sequence of the target loci (Abstract, Figure 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the barcodes of Pieprzyk to include the random and unique barcodes of McCloskey to arrive at the claimed invention with a reasonable expectation for success. Both Pieprzyk and McCloskey teach methods that include steps of barcoding, primers, amplification and sequencing. Pieprzyk and McCloskey use the barcodes within the method in different ways. Pieprzyk teaches “a different barcode primer can be employed to amplify one or more target sequences from each of a number of different samples, such that the barcode nucleotide sequence indicates the sample origin of the resulting amplicons” (paragraph 98, page 8). McCloskey teaches “the present invention provides methods for authenticating a nucleic acid molecule and its sequence with a molecular barcode and batch-stamp. In another aspect, the present invention provides methods for authenticating a nucleic acid amplification product” (Abstract). As an example, McCloskey also teaches “There were 22 sequences with a barcode that was identical to a sequence already obtained (i.e., redundant sequences). The remaining 110 sequences had distinct barcode regions that were 5 nucleotides long, indicating that those sequences originated from separate cells, or separate genomic target molecules” (paragraph 53). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the barcodes of Pieprzyk to include the random and different, individual barcodes of McCloskey to arrive at the claimed invention with a reasonable expectation for success.
Further, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of Pieprzyk to include the hybrid capture probes and enrichment as taught by Gnirke to arrive at the claimed invention with a reasonable expectation for success. Pieprzyk teaches a method of sequencing of cell-free nucleic acids using steps of analysis of cell free nucleic acids for amplification and sequencing. While Pieprzyk does not specifically teach the inclusion of hybrid capture probes, Gnirke teaches specific library based enrichment of nucleic acids with the inclusion of hybrid capture probes. Gnirke teaches “We developed a capture method that uses biotinylated RNA ‘baits’ to fish targets out of a ‘pond’ of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target 415,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out”. Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Pieprzyk to include the hybrid capture probes and enrichment as taught by Gnirke to arrive at the claimed invention with a reasonable expectation for success.
Claims 3-5 and 13-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over in Pieprzyk et al. (US PgPub 20140186827; July 2014), McCloskey et al. (PgPub 20070020640; January 2007) and Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189) as applied over claims 1-2, 6-12 and 16-21 and further in view of Rava et al. (US Patent 9,323,888; April 2016).
With regard to claim 3, Rava teaches a method of claim 1, wherein the plurality of target loci comprises between 100 and 2,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
With regard to claim 4, Rava teaches a method of claim 1, wherein the plurality of target loci comprises between 200 and 1,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
With regard to claim 5, Rava teaches a method of 1, wherein the plurality of target loci comprises between 300 and 2,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
With regard to claim 13, Rava teaches a method of claim 1, wherein the plurality of target loci comprises between 100 and 2,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
With regard to claim 14, Rava teaches a method of claim 1, wherein the plurality of target loci comprises between 200 and 1,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
With regard to claim 15, Rava teaches a method of 1, wherein the plurality of target loci comprises between 300 and 2,000 SNV loci (col. 37, lines 32-39, where 40 or more polymorphic sites are analyzed).
Rava teaches at least 40 polymorphic sites. Rava also suggests that 40 or more sites can be included.
An ordinary practitioner would have recognized that the results optimizable variables of time, product amount and number of loci which can be analyzed could be adjusted to maximize the desired results. As noted in In re Aller, 105 USPQ 233 at 235,
More particularly, where the general conditions
of a claim are disclosed in the prior art, it is
not inventive to discover the optimum or workable
ranges by routine experimentation.
Routine optimization is not considered inventive and no evidence has been presented that the number of sites detected and sequenced was other than routine, that the products resulting from the optimization have any unexpected properties, or that the results should be considered unexpected in any way as compared to the closest prior art.
Response to Arguments
Applicant's arguments filed August 21, 2025 have been fully considered but they are not persuasive.
Applicant traverses the rejection over Pieprzyk, McCloskey and Gnirke. Applicant also traverses the rejection further in view of Rava.
Applicant argues "Pieprzyk does not teach or suggest using high-throughput sequencing to obtain "sequence reads with a depth of read of at least 200 per target locus" as recited by the present claims.
The Office Action acknowledges at page 12 that Pieprzyk does not teach or suggest the "step wherein the extracted cell-free DNA from the biological sample is tagged with different
molecular barcodes." Moreover, Pieprzyk does not teach or suggest that "the molecular barcodes
in the sequence reads are used to identify sequence reads from the same isolated cell-free DNA
molecule and to measure a relative amount of each allele of each of the target loci in the single
biological sample" as recited by the present claims because Pieprzyk's sample barcodes cannot
distinguish between different DNA molecules from the same sample" (p 8 of remarks)
Applicant next argues "As stated in the title of Example 13, the method described in Example 13 and FIG. 11 is a "Target-Specific Super-Plexing" method. Moreover, paragraph [0071] states for both general procedures A and B that the disclosed method relies on using" 2 target specific oligos, Pl and P2, each bearing a different tag, to any contiguous nucleic acid" to produce "amplification of 10,000 separate amplicons." Accordingly, Example 13 does not teach or suggest "amplifying the plurality of barcoded DNA molecules by universal amplification to obtain a sequencing library" as recited by the present claims, but rather, discloses a "Target-Specific Super-Plexing" method" (p 9 of remarks)
Applicant describes other aspects of Pieprzyk and concludes "Pieprzyk uses target specific amplification in the referenced working examples 13 and 14, so Pieprzyk does not teach or suggest "amplifying the plurality of barcoded DNA molecules by universal amplification to obtain a sequencing library." (p 9 of remarks)
Applicant also claims there is a lack of motivation to combine references.
These arguments have been considered, but they are not persuasive.
Applicant's remarks regarding the teachings in the working examples were not persuasive as the argument omits key features of the passages cited. Applicant argues directly that “Example 13 does not teach or suggest "amplifying the plurality of barcoded DNA molecules by universal amplification to obtain a sequencing library" as recited by the present claims, but rather, discloses a "Target-Specific Super-Plexing" method" (p 9 of remarks).
Regarding Example 13, Applicant argues "As stated in the title of Example 13, the method described in Example 13 and FIG. 11 is a "Target-Specific Super-Plexing" method. Moreover, paragraph [0071] states for both general procedures A and B that the disclosed method relies on using" 2 target specific oligos, Pl and P2, each bearing a different tag, to any contiguous nucleic acid" to produce "amplification of 10,000 separate amplicons" (p 9 of remarks).
Except contrary to Applicant's arguments, Example 13 does specifically teach universal amplification. For example, within Example 13, at paragraph 672 Pieprzyk states "The Ligase Detection Reaction (LOR) or PCR in combination with target-specific oligos bearing 2 Universal Preamp target sites and target-specific tag sequences to ameliorate primer interaction issues observed in multiplex PCR. Addition of Universal Preamp priming sites permits the use of only two common primers to simultaneously multiplex preamp all ligation products. i.e. "Super-Plexing"" Therefore, contrary to the suggestion made in Applicant's remarks that Pierpzyk only teaches "target specific" superplexing, universal primer sites are a key part of the steps needed to make the super plexing functional. It is also noted that Pierpzyk uses the terminology of universal and common interchangeably and separately notes in paragraph 189, "if common nucleotide tags are employed, common tag-specific primers can be used to produce amplicons for detection. Such primers could introduce a binding site for a universal detection probe such that detection could be carried out using a single probe for multiple sequences". Stated another way, a common tag specific primer (or universal primer) can be used to produce amplicons. So, Pieprzyk incorporates steps of universal amplification in multiple ways. The universal amplification steps described by Pieprzyk may not be part of a "workflow" that exactly
matches the method as taught by the instant claims but it teaches and renders obvious, together with the teachings of other references, a method that includes steps of ligation of adaptor tags, performing universal amplification using these adaptor tags, enriching loci and sequencing these loci.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, both Pieprzyk and McCloskey are centered in the same field of study. Both of these references are focused on methods that include steps of barcoding, primers, amplification and sequencing. While these references may have some distinct steps within their method, the analysis and focus on specific tracking and analysis of sequences following amplification is shared between Pieprzyk and McCloskey. One of skill not only could but would look to references that share similar steps and approaches to methods of amplification, barcodes and sequencing and include those teachings together.
Therefore, while Applicant's arguments have been considered, they are not persuasive and the rejection is maintained. Further, as Applicant’s arguments regarding Pieprzyk and McCloskey are not persuasive, the rejection regarding Gnirke and Rava are also not persuasive and the rejection is maintained.
Citation of Pertinent Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Rava et al. (US 20110230358 A1; September 2011, IDS reference); Cuppens et al. (US 20100227329 A1; September 2010); Oliphant et al. (US 20030108900 A1; June 2003).
Conclusion
No claims are allowed. Claims 1-14 and 16-21 stand rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHANIE KANE MUMMERT whose telephone number is (571)272-8503. The examiner can normally be reached on M-F 9:00-5:30.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached on 571-272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/STEPHANIE K MUMMERT/Primary Examiner, Art Unit 1637
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