DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment filed on September 2, 2025 is acknowledged and has been entered. Claims 1 and 7 have been amended. Claims 17-18 have been added. Claims 1-16 are pending.
Claims 1-18 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 Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.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 as necessitated by Amendment.
New Grounds of Rejection necessitated by Amendment
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. 61448547, 61462972, 61426208, 61398159, 61395850, fails 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. While each of these priority documents include support for universal adaptors or universal amplification and mutation detection, none of these priority documents include sufficient (or any) support for the inclusion of barcodes or barcode primers, or the inclusion of cancer or tumor or that polymorphic loci are associated with tumor or cancer. Application 61516996 (‘996 application) does not provide support for depth of read as recited in the amendment to the claims or in claims 15-16.
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.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on March 14, 2025 and October 22, 2025 was filed in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 1 and 7 are objected to because of the following informalities: the amendment to step (iii) recites “produce a population of enrich plurality of loci” contains a typographical error. The amended phrase should read “enriched plurality of loci”. (emphasis added) Appropriate correction is required.
Previous Grounds of Rejection
Rejection updated to address amendment to the claims
Claim Interpretation
The term “depth of read” was not specifically defined and the connection between the term and the specific number was also not explained in the specification; however, the specification discusses the depth of read in sequencing is discussed at length. In the absence of additional detail, the amendment to the claim to include reference to “depth of read” in terms of a specific number is being interpreted as equivalent to thorough coverage of sequencing specific loci.
The amendment to the claim now recites “molecular barcode” and an additional step that recites “wherein the cell free DNA from the same blood sample is tagged with a plurality of different molecular barcodes”. The specification does not specifically define molecular barcodes, and does not use the particular language of “different molecular barcodes”. The specification, instead, states the molecular barcode “contains the randomly generated molecular barcode” (paragraph 513). Further, the specification describes “molecular barcoding primers may consist of a sequence that is not complementary to the target sequence followed by random molecular barcode region followed by a target specific sequence” (paragraph 500).
The claim language of molecular barcode, in the context of claim 1, will be interpreted as reading on a tag or barcode or label that includes a random sequence or other “unique” sequence that is either distinguishable, distinct or unique and is not used to label entire samples for pooling.
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.
Claim(s) 1-14 and 17-18 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), Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189) and Gormally et al. (Mutation Research, 2007, 635:105-117).
With regard to claim 1, Pieprzyk teaches a method for preparing a deoxyribonucleic acid (DNA) fraction from a cancer patient useful for analyzing one or more mutations of a cancer in a subject, comprising:
(a) collecting a blood sample from the subject;
(b) extracting cell free DNA molecules from the blood (paragraph 57, 172-173, 225, 713, where whole blood or maternal whole blood is used for extraction; see also pp 185, 191, 299, where the method is applicable to cancer patients)
(c) producing a fraction of the cell free DNA molecules extracted in (b) by
(i) ligating adaptor tags and a plurality of different molecular barcodes to the extracted cell free DNA molecules to generate a plurality of different barcoded DNA molecules (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);
(ii) performing universal amplification using the adaptor tags to produce a sequencing library from the plurality of barcoded DNA molecules (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);
(iii) enriching for a plurality of loci comprising 100-2,000 loci from the sequencing library (paragraph 70, which refers to Fig 10A-M and Example 12, where the method enriches for products for use within the method, as claimed; see also paragraph 644, within Example 12; see also paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed);
(d) performing sequencing on the enriched plurality of loci to obtain sequence reads for the plurality of loci (see for example, sequencing as described in Example 10, paragraph 632); and determine whether the plurality of loci comprise one or more mutations of the cancer based on the sequence reads obtained from sequencing (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632) wherein sequence reads from the same extracted cell-free DNA molecule are identified using the plurality of different 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; 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 plurality of loci comprises between 100 and 1,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
With regard to claim 3, Pieprzyk teaches a method of claim 1, wherein the plurality of loci comprises between 300 and 2,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
With regard to claim 4, Pieprzyk teaches a method of claim 1, wherein the molecular barcodes are not unique with respect to the cell free DNA to which they are attached (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 5, Pieprzyk teaches a method of claim 1, wherein the cell free DNA comprises mixed DNA from the cancer and from the host (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632; see also paragraph 493, where analysis of wild type to mutation is described).
With regard to claim 6, Pieprzyk teaches a method of claim 5, wherein the method further comprises determining the fraction of cell free DNA that is of cancer origin based on the sequence reads from the cancer DNA and the host DNA (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632; see also paragraph 493, where analysis of wild type to mutation is described).
With regard to claim 7, Pieprzyk teaches a method for preparing a deoxyribonucleic acid (DNA) fraction from a cancer patient useful for analyzing one or more mutations of a cancer in a subject, comprising:
(a) collecting a blood sample from the subject;
(b) extracting cell free DNA molecules from the blood sample (paragraph 57, 172-173, 225, 713, where whole blood or maternal whole blood is used for extraction; see also pp 185, 191, 299, where the method is applicable to cancer patients);
(c) producing a fraction of the cell free DNA molecules extracted in (b) by
(i) ligating adaptor tags and a plurality of different molecular barcodes to the extracted cell free DNA molecules to generate a plurality of barcoded DNA molecules (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);
(ii) performing a universal amplification using the adaptor tags to produce a sequencing library from the plurality of barcoded DNA molecules (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);
(iii) enriching a plurality of loci (paragraph 70, which refers to Fig 10A-M and Example 12, where the method enriches for products for use within the method, as claimed; see also paragraph 644, within Example 12; see also paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed);
(d) performing sequencing on the enriched plurality of loci to obtain sequence reads (see for example, sequencing as described in Example 10, paragraph 632); and determine whether the plurality of loci comprise one or more mutations of the cancer in the subject based on the sequence reads obtained from the sequencing (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632), wherein the plurality of loci comprises 300-2,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed) wherein sequence reads from the same extracted cell-free DNA molecule are identified using the plurality of different 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; 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 8, Pieprzyk teaches a method of claim 7, wherein the plurality of loci comprises between 300 and 1,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
With regard to claim 9, Pieprzyk teaches a method of claim 7, wherein the method further comprises determining mutations in the plurality of loci based on the sequence reads (paragraph 658, for example, where a mutation is detected).
With regard to claim 10, Pieprzyk teaches a method of claim 7, wherein barcoded DNA from each targeted locus have a unique molecular barcode (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 of claim 9, wherein the method further comprises determining the number of unique molecules in the blood sample for each locus based on sequence reads from the molecular barcodes and the cell free DNA (paragraph 228, gives an example of how a unique molecule can be identified within a large volume of loci or targets).
With regard to claim 12, Pieprzyk teaches a method of claim 7, wherein the molecular barcodes are not unique with respect to the cell free DNA to which they are attached (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 13, Pieprzyk teaches a method of claim 7, wherein the cell free DNA comprises mixed DNA from the cancer and from the host (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632; see also paragraph 493, where analysis of wild type to mutation is described).
With regard to claim 14, Pieprzyk teaches a method of claim 13, wherein the method further comprises determining the fraction of cell free DNA that is of cancer origin based on the sequence reads from the cancer DNA and the host DNA (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632; see also paragraph 493, where analysis of wild type to mutation is described).
Regarding claim 1, while Pieprzyk teaches the inclusion of barcodes, Pieprzyk does not teach the step wherein the nucleic acids from the same biological sample are tagged with different molecular barcodes.
With regard to claims 1 and 7, McCloskey teaches wherein the plurality of barcoded DNA from the same blood sample are tagged with at least a subset of the 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 17, McCloskey teaches a method of claim 1, wherein the plurality of molecular barcodes comprises at least 1024 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).
With regard to claim 18, McCloskey teaches a method of claim 7, wherein the plurality of molecular barcodes comprises at least 1024 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).
Regarding claims 1 and 7, while Pieprzyk teaches enrichment of short nucleic acids, Pieprzyk does not teach the use of hybrid capture probes. Further, while Pieprzyk teaches sequencing, Pieprzyk is not specific regarding massively parallel sequencing.
Regarding claim 1, while Pieprzyk teaches the method as claimed, Pieprzyk does not teach performing massively parallel sequencing on the enriched plurality of loci to obtain sequence reads with a depth of read of at least 200 per target locus.
Regarding claim 7, does not teach performing massively parallel sequencing on the enriched plurality of loci to obtain sequence reads with a depth of read of at least 200 per target locus.
With regard to claim 1 and 7, Gnirke teaches
(iii) enriching for a plurality of loci comprising 100-2,000 loci from the sequencing library using hybrid capture probes that target the plurality of loci to produce a population of enrich plurality of loci (Abstract, Figure 1, where hybrid capture probe enrichment is described); and
(i) performing massively parallel sequencing on the population of enriched plurality of loci to obtain sequence reads for the plurality of loci (p. 186, col. 2 “Discussion” heading; p 188, “Catch processing and sequencing” heading) and
(ii) determining whether the plurality of loci comprise one or more mutations based on the sequence reads obtained from the massively parallel sequencing (p. 186, col. 2 “Discussion” heading; p 188, “Catch processing and sequencing” heading);
(d) performing sequencing on the population of enriched plurality of loci to obtain sequence reads with a depth of read of at least 200 per target locus (p 185, col. 1, under heading “Regional Capture and Sequencing” which states “The average depth of coverage for the 0.75 million genome bases covered by bait in the four target regions was 221”; see also for example, sequencing as described in Example 10, paragraph 632); and determine whether the plurality of loci comprise one or more mutations of the cancer in the subject based on the sequence reads obtained from the sequencing (paragraph 658, for example, where a mutation is detected; see for example, sequencing as described in Example 10, paragraph 632), wherein the plurality of loci comprises 300-2,000 loci (paragraph 183, for example, where a plurality up to 1000 target loci can be analyzed).
Regarding claims 1 and 7, while Gnirke teaches massively parallel sequencing, neither Pieprzyk or Gnirke are specific in detecting cancer specific mutations using the method, as claimed.
With regard to claims 1, 5-7, 13 and 14, Gormally teaches methods that would include:
(ii) determining whether the plurality of loci comprise one or more mutations of the cancer based on the sequence (Fig 1, p 107-108, where the role of cell free DNA in mutation detection is generally described; Table 3, p 112, col. 1, top of page).
With regard to claim 5, Gormally teaches a method of claim 1, wherein the DNA comprises mixed DNA from the cancer and from the host (Fig 1, p 107-108, where the role of cell free DNA in mutation detection is generally described; Table 3, p 112, col. 1, top of page).
With regard to claim 6, Gormally teaches a method of claim 5, wherein the method further comprises determining the fraction of DNA that is of cancer origin based on the sequence reads from the cancer DNA and the host DNA (Fig 1, p 107-108, where the role of cell free DNA in mutation detection is generally described; Table 3, p 112, col. 1, top of page; see also p 112, “4.2 CFDNA in case-control studies” heading, where tumoral origin in CFDNA samples is described).
With regard to claim 13, Gormally teaches a method of claim 7, wherein the DNA comprises mixed DNA from the cancer and from the host (Fig 1, p 107-108, where the role of cell free DNA in mutation detection is generally described; Table 3, p 112, col. 1, top of page; see also p 112, “4.2 CFDNA in case-control studies” heading, where tumoral origin in CFDNA samples is described).
With regard to claim 14, Gormally teaches a method of claim 13, wherein the method further comprises determining the fraction of DNA that is of cancer origin based on the sequence reads from the cancer DNA and the host DNA (Fig 1, p 107-108, where the role of cell free DNA in mutation detection is generally described; Table 3, p 112, col. 1, top of page; see also p 112, “4.2 CFDNA in case-control studies” heading, where tumoral origin in CFDNA samples is described).
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 different, individual 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, enrichment and massively parallel sequencing techniques 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”. As noted in that statement, Gnirke also focuses on massively parallel sequencing and also notes “With further optimization, routine implementation of hybrid selection would enable deep, targeted next-generation sequencing of thousands of exons as well as of megabase-sized candidate regions implicated by genetic screens. Targeting based on hybrid selection may be potentially useful for a variety of other applications as well, where traditional singleplex PCR is either too costly or too specific in that specific primers may fail to produce a PCR product that represents all genetic variation in the sample. Examples are enrichment of precious ancient DNA that is heavily contaminated with unwanted DNA, deep sequencing of viral populations in clinical samples, or metagenomic analyses of environmental or medical specimens” (p. 186, col. 2 “Discussion” heading). 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, enrichment and massively parallel sequencing as taught by Gnirke to arrive at the claimed invention with a reasonable expectation for success.
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 and Gnirke to apply the method to detection of cancer as described by Gormally to arrive at the claimed invention with a reasonable expectation for success. First, Pieprzyk specifically teaches the method could be useful in cancer, “These enrichment/selective tagging methods can be combined with methods described above to further facilitate the detection and or quantification of target sequences in samples having mixed length nucleic acids (e.g. fetal DNA in maternal plasma or tumor DNA in plasma from cancer patients.”. Pieprzyk also notes “These methods can also be employed in determinations DNA or RNA copy number. Determinations of aberrant DNA copy number in genomic DNA is useful, for example, in the diagnosis and/or prognosis of genetic defects and diseases, such as cancer” (paragraph 299). These methods can be carried out, for example, to determine a fetal genotype or determine the presence of a mutation or fetal aneuploidy” (paragraph 60). Pieprzyk also teaches “These methods can be carried out, for example, to determine a fetal genotype or determine the presence of a mutation or fetal aneuploidy” (paragraph 176). Next, Gormally teaches “the technical issues involved in obtaining, using and analyzing CFDNA in cancer or healthy subjects.We also summarize the literature available on the mechanisms of CDNA release as well as on cross-sectional or prospective studies aimed at assessing the clinical and biological significance of CFDNA. These studies show that, in some circumstances, CFDNA alterations are detectable ahead of cancer diagnosis, raising the possibility of exploiting them as biomarkers for monitoring cancer occurrence” (Abstract). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Pieprzyk and Gnirke to apply the method to detection of cancer as described by Gormally to arrive at the claimed invention with a reasonable expectation for success.
Claim(s) 15-16 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), Gnirke et al. (Nature Biotechnology, 2009, 27(2):182-189), Gormally et al. (Mutation Research, 2007, 635:105-117) as applied over claims 1-14 and further in view of Li et al. (Genome Research, 2009, 19:1124-1132).
Regarding claim 1, while Pieprzyk teaches the method as claimed, Pieprzyk does not teach performing massively parallel sequencing on the enriched plurality of loci to obtain sequence reads with a depth of read of at least 200 per target locus.
Regarding claim 7, does not teach performing massively parallel sequencing on the enriched plurality of loci to obtain sequence reads with a depth of read of at least 200 per target locus.
With regard to claim 15, Li teaches a method of claim 1, wherein the depth of read of is at least 10,000 per target locus (Abstract, p. 1129, col. 2, Table 3, where the high depth of read is described in more detail).
With regard to claim 16, Li teaches a method of claim 7, wherein the depth of read of is at least 10,000 per target locus (Abstract, p. 1129, col. 2, Table 3, where the high depth of read is described in more detail).
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 massively parallel sequencing techniques as taught by Gnirke and to incorporate depth of read as described by Li 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. Gnirke focuses on massively parallel sequencing and also notes “With further optimization, routine implementation of hybrid selection would enable deep, targeted next-generation sequencing of thousands of exons as well as of megabase-sized candidate regions implicated by genetic screens. Targeting based on hybrid selection may be potentially useful for a variety of other applications as well, where traditional singleplex PCR is either too costly or too specific in that specific primers may fail to produce a PCR product that represents all genetic variation in the sample. Examples are enrichment of precious ancient DNA that is heavily contaminated with unwanted DNA, deep sequencing of viral populations in clinical samples, or metagenomic analyses of environmental or medical specimens” (p. 186, col. 2 “Discussion” heading). While Gnirke discusses sequencing depth, Gnirke does not focus specifically on depth of read. Li teaches “SNP detection accuracy is related to the sequencing error rate and read length. In the Asian genome sequence, the error rate over all the mappable reads was ;1.4%, and read length averaged 35 bp. We have recently been able to reduce this error rate to 0.5%;0.8% in reads of 35 bp and to 0.5%;1.5% for 50;75-bp read length in a typical run, and thus expect to be able obtain very accurate SNP calling from low-depth (such as 4;103) paired-end sequencing in the near future with continuous improvement in data quality”. Li also teaches the method “showed that using dbSNP genotypes for prior probability calculation substantially helps in distinguishing real heterozygotes from errors in regions of low-depth sequencing. The use of additional information for prior probability under the general Bayesian probability framework could likely aid in further improving accuracy of posterior probability calculation” (p. 1130, col. 2). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Pieprzyk to include massively parallel sequencing techniques as taught by Gnirke and to incorporate depth of read as described by Li to arrive at the claimed invention with a reasonable expectation for success.
Citation of Pertinent Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Maughan et al. (The Plant Genome, 2009, 2(3):260-270). Cummings et al. (BMC Genomics, 2010:11, 641, p 1-9). Anker et al. (Cancer and Metastasis Reviews, 1999, 18:65-73).
Response to Arguments
Applicant traverses the rejection over Pieprzyk, McCloskey and Gnirke and Gormally. Applicant also traverses the rejection further in view of Li.
Applicant argues "Pieprzyk and McCloskey do not teach or suggest using "the barcodes
within the method in different ways" as alleged in the Office Action at page 13. Pieprzyk teaches
using barcodes as acknowledged in the Office Action at page 14 to indicate "sample origin of the
resulting amplicons" as explained at paragraph [0098]" (p 8 of remarks).
Applicant argues McCloskey does not cure the deficiencies of the other references.
Applicant argues "McCloskey does not teach or suggest using a plurality of different molecular barcodes the same way as recited by the present claims". Applicant also argues "McCloskey teaches methods for authenticating an amplification product by "contacting a target nucleic acid molecule in a sample with a bar-coded oligonucleotide under suitable conditions to anneal the bar-coded oligonucleotide to the target nucleic acid molecule" and that McCloskey uses barcodes "to tag a single specific target nucleic acid" (p 8 of remarks). Applicant also argues McCloskey is used to tag "barcoded FMRl products" and that McCloskey "teaches bar-coding a gene specific region (FMRl), and only molecules containing a FMRl target sequence will get tagged" (p 9 of remarks).
Applicant concludes "McCloskey does not teach or suggest identifying "sequence reads from the same isolated cell-free DNA molecule" by using the molecular barcodes" (p 9 of remarks).
Next, Applicant argues there is a lack of motivation to combine Pieprzyk and McCloskey. Applicant argues "Pieprzyk refers to sample barcodes for sample multiplexing and
sample origin determination and does not need to use molecular barcodes, whereas McCloskey
teaches using barcoding for authenticating an amplification product of a single target gene" (p 8
of remarks). Applicant argues at length regarding the case law underpinning the motivation to
combine and the need to identify a reason for a combination of references (p 8-9 of remarks).
Applicant concludes that Gnirke, Gormally and Li cannot cure the deficiencies of Pieprzyk and
McCloskey and that the combination of references "fail to teach or suggest each and every
element of the claimed invention" (p 9-10 of remarks).
These arguments have been considered, but they are not persuasive.
First, it is noted that the primary aspect of the rejection that teaches the steps of
sequencing a sample and using sequence reads, as claimed, is Pieprzyk, except Applicant's
remarks are mostly aimed at McCloskey, instead. It is also noted, as Applicant even
acknowledges in their arguments, Pieprzyk is focused on identification of the origin of a
sequence. The only distinction between the teachings of Pieprzyk and the method as claimed is
that Pieprzyk does not teach plural or different barcodes. While McCloskey may not teach the
barcodes exactly in the same manner as the method as claimed, and as amended, McCloskey
without question teaches "a plurality of different barcodes".
While Applicant's remarks regarding Pieprzyk are centered on an emphasis on sample
specific origin, for example, Pieprzyk teaches the method can be focused on identification in a
variety of contexts. Pieprzyk teaches "The methods of the invention are applicable to any
technique aimed at 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, such as amplifications, deletions, or aneuploidy. The methods may be employed
in genotyping, which can be carried out in a number of contexts, including diagnosis of genetic
diseases or disorders, pharmacogenomics (personalized medicine), quality control in agriculture (
e.g., for seeds or livestock), the study and management of populations of plants or animals (e.g.,
in aquaculture or fisheries management or in the determination of population diversity), or
paternity or forensic identifications. The methods of the invention can be applied in the
identification of sequences indicative of particular conditions or organisms in biological or
environmental samples" (pp 298). Further, as established in the rejection of record, at Example
10, which describes a "method of fetal aneuploidy detection by next generation sequencers as published by both Quake and Lo", a "multiplex reaction is performed and then run on the
sequencer. They both create reactions products that can be identified and counted to make the
determination of aneuploidy" (pp 626-632). While Pieprzyk incorporates molecular barcodes in
the method of aneuploidy determination, identification of specific sequences is a key part of the
method as described by Pieprzyk.
Furthermore, regarding the assertion that McCloskey is only useful for authentication of
amplification or that only a single target is tagged, contrary to Applicant's arguments, and as the
rejection clearly states, McCloskey includes 1000+ different, distinct barcodes. Further, while
Applicant argues McCloskey "fails to teach or suggest and that sequence reads derived from the
same original cell-free DNA molecule are identified using the molecular barcode," as recited by
the present claims, in fact, on this subject, McCloskey specifically teaches "'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. (paragraph 21 ). Further, regarding the issue of identification, McCloskey teaches "Each genomic fragment is marked prior to amplification, allowing us to identify contaminant and redundant sequences and to quantify accurately the proportion of cells carrying a particular sequence variant by counting only distinctly tagged sequences". Just like the instantly claimed method is focused on a method
where sequence reads derived from the same original cell-free DNA molecule are identified
using the molecular barcode", McCloskey is focused on counting only uniquely tagged sequence
variants.
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., 5 50 U.S. 3 98, 82 USPQ2d
13 85 (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, Gormally and Li are also not persuasive and the rejection is maintained.
Conclusion
No claims are allowed. All claims stand rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHANIE KANE MUMMERT whose telephone number is (571)272-8503. The examiner can normally be reached M-F 9:00-5:30.
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/STEPHANIE K MUMMERT/Primary Examiner, Art Unit 1637
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