DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 01/20/2026 has been entered.
Claims Status
Claims 1-7, 14, 15, 17, 18, 21, 22, & 24-28 filed on 01/20/2026 are pending. Claims 7, 22, & 24 are currently under examination directed to the elected species single nucleotide variant (SNV), the genomic sequence of EGFR, and breast cancer, respectively and the species of TP53, KRAS, and APC are rejoined with EFGR in claim 22 and the species of prostate cancer is rejoined with breast cancer in claim 24 (see response dated 01/21/2025). All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections are either newly applied, as necessitated by amendment, or are reiterated. They constitute the complete set being presently applied to the instant application. Response to Applicant’s argument follow. This action is Non-FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Any rejection not reiterated is hereby withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 112
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 1-7, 14, 15, 17, 18, 21, 22, & 24-28 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.
Regarding claims 1 & 2, claims 1 & 2 have been amended to recite “whereby each grouped family consists of reads of the same target sequence, reads of the same fragment length, and one or more reads with a different UMI sequence”. The specification of the instant application has been thoroughly reviewed by support for this newly added limitation was not found and the reply filed on 01/20/2026 generally states “the amendments are fully supported by the original specification, examples, and claims” and does not provide citations to where there is support for this amendment in the instant application. Further, while there is no in haec verba requirement, newly added claims or claim limitations must be supported in the specification through express, implicit, or inherent disclosure. In the instant situation, this limitation of whereby each grouped family consists of one or more reads with a different UMI sequence is not expressly recited in the specification of the instant application. Additionally, there does not appear to be implicit or inherent support for this limitation of grouped read family of one or more reads with a different UMI sequence because the specification of the instant application discusses “grouping sequencing reads based on fragment size and UMI into read families; requiring consensus among all sequencing reads in a read family” and “in which each UMI is a node … all of the reads from UMIs in each component from the resulting graph constitute a read family and are considered to have come from the same original molecule” (pg. 5 & 28 of the instant specification). Since it is not clear how the newly added limitations are implicitly or inherently disclosed, the claims are rejected under 35 USC 112(a). In response, applicant may cancel new matter, or provide explanations as to where the limitations find express, implicit, or inherent support.
Claims 5-7, 14, 15, 17, 18, 21, 22, & 24-27 are rejected due to this dependence on claim 1 and claims 3, 4, & 28 are rejected due to their dependence on claim 2.
Claim Rejections - 35 USC § 103
Claim(s) 1-7, 14, 15, 18, 22, & 24-26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Talasaz (U.S. Patent Application Publication No. 2018/0023125), as cited on the IDS dated 02/27/2024, in view of Murtaza (U.S. Patent Application Publication No. 2019/0292575) and Jee (Jee et al.; Nature Letter, Vol. 534, pages 693-708, June 2016).
Regarding amended claim 1, Talasaz teaches a method for detecting genetic aberrations in cfDNA from a subject through providing cfDNA obtained from a bodily sample of the subject, which can be obtained from blood from the subject (paragraph [0225] lines 4-6), in which the initial starting genetic material can be enriched (paragraph [0055] lines 1-6), then tags comprising barcodes are attached (ligated) to the cfDNA molecules through blunt end ligation to both ends of the cfDNA molecules (comprising ligation of an adapter to the 3’ end) (pg. 34 column 2 claim 9 lines 1-2; pg. 34 column 2 claim 11 lines 1-3), followed by amplifying the tagged parent polynucleotides to produce tagged progeny polynucleotides (nested second target-specific primers to produce progeny polynucleotides with sample barcodes), sequencing the tagged progeny polynucleotides to produce sequence reads wherein each sequence read comprises a barcode sequence and a sequence derived from a cfDNA molecule, grouping the sequence reads into families (read families) based on the barcode sequence, comparing the sequence reads grouped within each family to determine consensus sequences for each family, and then detecting at one or more genetic loci a plurality of genetic aberrations (detecting presence or absence of one or more genetic variants) (pg. 34 column 1 claim 1 lines 1-26). Talasaz also teaches grouping sequence reads of unique identity based on sequence information at the beginning (start) and end (stop) regions of the sequence reads, the length of the sequence read (fragment size), and the attachment of a barcode (UMI) and grouping, comparing, and then determining consensus sequences for each family and that detecting copy number variation (genetic aberration) of consensus sequences between at least two sets of parent polynucleotides (a true mutation requires that the mutation is detected in all sequencing reads for at least two independent read families) (paragraph [0100] lines 4-8; paragraph [0124] lines 1-10; pg. 34 column 1 claim 1 lines 1-26). Talasaz also teaches that each polynucleotide in a set is mappable to a reference sequence (paragraph [0115] lines 1-5). Talasaz also teaches that this method can be used to modify therapy based on the most recent sample analysis in which the method taught by Talasaz allows for sample analysis for inferring the genetic profile of a tumor, infection, or other tissue abnormality allowing for the monitoring and treatment modification of that sample (paragraph [0030] lines 1-6). Finally, Talasaz teaches a blood sample may be taken from a prostate cancer subject and then processed according to the method taught above in which the sequence data is assembled and analyzed for copy number variation through mapping to a healthy individual (reference genome) and that this data can indicate if the subject’s cancer have progressed and is resistant to treatment which then would require the prescription of a new treatment ([0335] lines 1-5; paragraph [0337] lines 1-7; paragraph [0340] lines 1-12).
Talasaz fails to teach linearly amplifying the cfDNA with a first target-specific primers and that the adapter oligonucleotide comprises a stem-loop intramolecular nucleotide base pairing, a hydroxyl group at the 3'-end; a phosphate at the 5'-end; a random region complementary to the nucleic acid sequence; and a random region in the loop comprising a unique molecular identifier (UMI) and whereby each grouped family consists of reads with one or more different UMI sequence.
Murtaza teaches a method for adding an oligonucleotide tag to a nucleic acid sequence through linearly amplifying the nucleic acid sequence and ligating an adapter oligonucleotide to the 3’-end of the nucleic acid sequence wherein the adapter oligonucleotide sequence comprises a stem-loop intramolecular nucleotide base pairing, a hydroxyl group at the 3'-end; a phosphate at the 5'-end; a random region complementary to the nucleic acid sequence; and a random region in the loop comprising a molecular barcode (unique molecular identifier) in which prior to ligation of an adapter oligonucleotide the sample may be linearly pre-amplified with a first primer complementary to a sequence of interest to increase the sample number of a biological cfDNA sample (linearly amplifying cfDNA with first target-specific primers complementary to the target sequence) (paragraph [0008] lines 1-8; paragraph [0011] lines 1-16; paragraph [0014] lines 1-8; paragraph [0019] lines 1-4; paragraph [0042] lines 5-9; pg. 8 column 1 claim 1 lines 1-17; pg. 8 column 2 claim 19 lines 1-3). Murtaza also teaches that molecular barcoding or tagging in the first or early steps of sequencing library preparation allows for the distinguishment of low abundance signals of variant for noise introduced during the process and that this process is a viable approach for the detection and quantification of low abundance variants in a complex mixture of nucleic acids which can be beneficial to the early detection of cancer (paragraph [0004] lines 1-18).
Jee teaches a method of attaching a barcode to target genomic DNA, linearly amplifying, attaching a second barcode, exponentially amplifying, sequencing, and then grouping the reads by both the primary barcode (first barcode corresponding to a read family) and the secondary barcode corresponding to a subfamily of reads (secondary different barcode corresponding to a read subfamily) with the same parent from a particular linear amplification step (each grouped read family consists of reads of one or more reads with a different UMI sequence) (pg. 697 column 1 2nd full paragraph lines 1-31; Extended Data Figure 2). Jee also teaches that the method of attaching and grouping read families with two different barcodes enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis (pg. 697 column 1 2nd full paragraph lines 15-21).
Talasaz, Murtaza, and Jee are considered to be analogous to the claimed invention because they are all in the same field of detection of amplification and sequencing of variant targets. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of ligating an adapter to the cfDNA molecules as taught in Talasaz to incorporate the use of the specific adapter oligonucleotide and linearly pre-amplifying before ligation of the specific adapter oligonucleotide as taught in Murtaza because Murtaza teaches that doing so would provide a viable method for the detection and quantification of low abundance variants and it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of grouping sequence reads of unique identity based on sequence information at the beginning (start) and end (stop) regions of the sequence reads, the length of the sequence read (fragment size), and the attachment of a barcode (UMI) as taught in Talasaz to incorporate the grouping the reads by both a primary barcode and the secondary barcode corresponding to a subfamily of reads (each grouped read family consists of reads of one or more reads with a different UMI sequence) as taught in Jee because Jee teaches that doing so would enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis.
Regarding amended claim 2, Talasaz teaches a method for detecting genetic aberrations in cfDNA from a subject through providing cfDNA obtained from a bodily sample of the subject, which can be obtained from blood from the subject (paragraph [0225] lines 4-6), in which the initial starting genetic material can be enriched (paragraph [0055] lines 1-6), then tags comprising barcodes are attached (ligated) to the cfDNA molecules through blunt end ligation to both ends of the cfDNA molecules (comprising ligation of an adapter to the 3’ end) (pg. 34 column 2 claim 9 lines 1-2; pg. 34 column 2 claim 11 lines 1-3), followed by amplifying the tagged parent polynucleotides to produce tagged progeny polynucleotides (nested second target-specific primers to produce progeny polynucleotides with sample barcodes), sequencing the tagged progeny polynucleotides to produce sequence reads wherein each sequence read comprises a barcode sequence and a sequence derived from a cfDNA molecule, grouping the sequence reads into families (read families) based on the barcode sequence, comparing the sequence reads grouped within each family to determine consensus sequences for each family, and then detecting at one or more genetic loci a plurality of genetic aberrations (detecting presence or absence of one or more genetic variants) (pg. 34 column 1 claim 1 lines 1-26). Talasaz also teaches grouping sequence reads of unique identity based on sequence information at the beginning (start) and end (stop) regions of the sequence reads, the length of the sequence read (fragment size), and the attachment of a barcode (UMI) and grouping, comparing, and then determining consensus sequences for each family and that detecting copy number variation (genetic aberration) of consensus sequences between at least two sets of parent polynucleotides (a true mutation requires that the mutation is detected in all sequencing reads for at least two independent read families) (paragraph [0100] lines 4-8; paragraph [0124] lines 1-10; pg. 34 column 1 claim 1 lines 1-26). Talasaz also teaches that each polynucleotide in a set is mappable to a reference sequence (paragraph [0115] lines 1-5). Talasaz also teaches that this method can be used to modify therapy based on the most recent sample analysis in which the method taught by Talasaz allows for sample analysis for inferring the genetic profile of a tumor, infection, or other tissue abnormality allowing for the monitoring and treatment modification of that sample (paragraph [0030] lines 1-6). Finally, Talasaz teaches a blood sample may be taken from a prostate cancer subject and then processed according to the method taught above in which the sequence data is assembled and analyzed for copy number variation through mapping to a healthy individual (reference genome) and that this data can indicate if the subject’s cancer have progressed and is resistant to treatment which then would require the prescription of a new treatment ([0335] lines 1-5; paragraph [0337] lines 1-7; paragraph [0340] lines 1-12).
Talasaz fails to teach linearly amplifying the cfDNA with a first target-specific primers and that the adapter oligonucleotide comprises a stem-loop intramolecular nucleotide base pairing, a hydroxyl group at the 3'-end; a phosphate at the 5'-end; a random region complementary to the nucleic acid sequence; and a random region in the loop comprising a unique molecular identifier (UMI) and whereby each grouped family consists of reads with one or more different UMI sequence.
Murtaza teaches a method for adding an oligonucleotide tag to a nucleic acid sequence through linearly amplifying the nucleic acid sequence and ligating an adapter oligonucleotide to the 3’-end of the nucleic acid sequence wherein the adapter oligonucleotide sequence comprises a stem-loop intramolecular nucleotide base pairing, a hydroxyl group at the 3'-end; a phosphate at the 5'-end; a random region complementary to the nucleic acid sequence; and a random region in the loop comprising a molecular barcode (unique molecular identifier) in which prior to ligation of an adapter oligonucleotide the sample may be linearly pre-amplified with a first primer complementary to a sequence of interest to increase the sample number of a biological cfDNA sample (linearly amplifying cfDNA with first target-specific primers complementary to the target sequence) (paragraph [0008] lines 1-8; paragraph [0011] lines 1-16; paragraph [0014] lines 1-8; paragraph [0019] lines 1-4; paragraph [0042] lines 5-9; pg. 8 column 1 claim 1 lines 1-17; pg. 8 column 2 claim 19 lines 1-3). Murtaza also teaches that molecular barcoding or tagging in the first or early steps of sequencing library preparation allows for the distinguishment of low abundance signals of variant for noise introduced during the process and that this process is a viable approach for the detection and quantification of low abundance variants in a complex mixture of nucleic acids which can be beneficial to the early detection of cancer (paragraph [0004] lines 1-18).
Jee teaches a method of attaching a barcode to target genomic DNA, linearly amplifying, attaching a second barcode, exponentially amplifying, sequencing, and then grouping the reads by both the primary barcode (first barcode corresponding to a read family) and the secondary barcode corresponding to a subfamily of reads (secondary different barcode corresponding to a read subfamily) with the same parent from a particular linear amplification step (each grouped read family consists of reads of one or more reads with a different UMI sequence) (pg. 697 column 1 2nd full paragraph lines 1-31; Extended Data Figure 2). Jee also teaches that the method of attaching and grouping read families with two different barcodes enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis (pg. 697 column 1 2nd full paragraph lines 15-21).
Talasaz, Murtaza, and Jee are considered to be analogous to the claimed invention because they are all in the same field of detection of amplification and sequencing of variant targets. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of ligating an adapter to the cfDNA molecules taught in Talasaz to incorporate the use of the specific adapter oligonucleotide and linearly pre-amplifying before ligation of the specific adapter oligonucleotide as taught in Murtaza because Murtaza teaches that doing so would provide a viable method for the detection and quantification of low abundance variants and it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of grouping sequence reads of unique identity based on sequence information at the beginning (start) and end (stop) regions of the sequence reads, the length of the sequence read (fragment size), and the attachment of a barcode (UMI) as taught in Talasaz to incorporate the grouping the reads by both a primary barcode and the secondary barcode corresponding to a subfamily of reads (each grouped read family consists of reads of one or more reads with a different UMI sequence) as taught in Jee because Jee teaches that doing so would enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis.
Regarding claims 3 & 4, Talasaz teaches generating a report of mapping genome positions and copy number variations from the cfDNA blood sample taken from a subject with prostate cancer to a healthy individual to determine the copy number variation profile of the cancer sample in which this analysis can provide information on progression of cancer and/or indication for the need to recommend a new treatment for the patient from which the cancer sample was taken (generating a report that includes a cell-free tumor mutation profile of the patient based on the detection of the presence or absence of the one or more somatic genetic variants) (paragraph [0335] lines 1-5; paragraph [0337] lines 1-7; paragraph [0339] lines 1-3 paragraph [0340] lines 1-12).
Regarding claim 5, Talasaz teaches a cfDNA sample taken from a stage II prostate cancer sample (paragraph [0335] lines 1-5; paragraph [0340] lines 1-12) and that the blood sample comprises less than 10 ng of cfDNA (pg. 34 column 1 claim 1 lines 1-26; pg. 34 column 1 claim 3 lines 1-2). Talasaz also teaches that this method may be useful in monitoring residual disease or recurrence of disease (paragraph [00314] lines 12-13).
Regarding claim 6, Talasaz teaches that the methods comprises the use of genetic data obtained from a tissue (germline DNA) or tumor biopsy from the sample patient (whole genome of tumor biopsy DNA) (paragraph [0032] lines 1-3).
Regarding claim 7, Talasaz teaches a method for detecting genetic aberrations which comprise single nucleotide variants (paragraph [0057] lines 1-6).
Regarding claim 14, Talasaz teaches sequencing reads are mapped and compared to a healthy individual in which the sequence reads (part of the read families) are compared to one another and a frequency of variant bases may be calculated as the number of reads containing the variant divided by the total number of reads and may be expresses as a ratio for each mappable position in which this frequency can range from 5% to 100% (ratio of 0.05 to 1) (mutant RFs divided by total RFs is at least 0.5) (paragraph [0301] lines 1-6; paragraph [0315] lines 1-6; paragraph [0344] lines 1-6).
Regarding claim 15, Talasaz teaches grouping the sequence reads into families (clusters) based on the barcode sequence, comparing the sequence reads grouped within each family to determine consensus sequences for each family, and then detecting at one or more genetic loci a plurality of genetic aberrations (RFs covering a targeted genomic locus are sorted by members in the RF) (pg. 34 column 1 claim 1 lines 1-26)
Regarding claim 18, Talasaz teaches methods for reducing or eliminating errors introduced by amplification and/or sequencing error by grouping sequence reads into families (RFs) and determining frequencies of base calls based on probabilities derived from family information (paragraph [0207] lines 1-15; paragraph [0210] lines 1-6 & 10-12 & 14-23; paragraph [0211] lines).
Regarding claim 22, Talasaz teaches selectively enriching polynucleotides (amplifying target regions) mapping to a gene selected from a group consisting of KRAS, APC, and TP53 (pg. 34 column 2 claim 16 lines 1-4).
Regarding claim 24, Talasaz teaches analyzing a prostate cancer sample (paragraph [0335] lines 1-5; paragraph [0340] lines 1-12).
Regarding claim 25, Talasaz teaches the collection a sample from a prostate cancer subject after completion of chemotherapy and radiation (completion of an anti-cancer therapy) to monitor remission of the subject in which at month 18 (method is repeated for more than one blood sample multiple days after the completion of an anti-cancer therapy) a blood sample is taken from the subject and determined to have a cell-free tumor burden of 5% leading to the prescription of treatment again (paragraph [0342] lines 1-6; paragraph [0344] lines 1-7; paragraph [0348] lines 1-5).
Regarding claim 26, Talasaz teaches the patient is a prostate cancer patient that has been treated with chemotherapy (neoadjuvant therapy) (paragraph [0342] lines 1-3).
Claim(s) 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Talasaz (U.S. Patent Application Publication No. 2018/0023125), as cited on the IDS dated 02/27/2024, and Murtaza (U.S. Patent Application Publication No. 2019/0292575) and Jee (Jee et al.; Nature Letter, Vol. 534, pages 693-708, June 2016), as applied to claims 1-7, 14, 15, 18, 22, & 24-26, and further in view of Liu (Liu, He, & Sun; PLOS Genetics, Vol. 14, pages 1-18, November 2018).
The teachings of Talasaz, Murtaza, and Jee with respect to claim 1 is discussed above.
Regarding claim 17, Talasaz teaches that collapsing sequence reads into families further comprises determining the quantitative measure of sequence reads in each family and that those consensus sequences are mapped to a particular reference location to detect instances of genetic variation through identifying areas in the genome in which copy number varies (paragraph [0266] lines 1-10) and that determining this measure of unique tagged polynucleotides can be performed using statistical or probabilistic models (paragraph [0149] lines 8-10).
Talasaz and Jee fails to teach applying multiple testing correction using the Bonferroni approach.
Liu teaches a method for assessing associations between somatic mutations and gene expression in 12 cancer types and that for the mutation-level association analysis between 37 mutations the Bonferroni correction was adopted for multiple testing correction and the corrected p-value less than 0.05 was required (pg. 1 abstract lines 16-17; pg. 8 & 9 paragraph bridging pg. 8 & pg. 9 lines 1-5). Liu also teaches that understanding associations between somatic mutations and cancer-related traits is important for precision cancer therapy and that this method provides a statistically powerful and computationally efficient approach for the association analysis of somatic mutations while also accounting for measurement errors of somatic mutations (pg. 13 3rd full paragraph lines 1-7).
Talasaz, Murtaza, Jee, and Liu are considered to be analogous to the claimed invention because they are all in the same field of detection of somatic mutations with cfDNA samples. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detection of genetic aberrations in a cfDNA sample taught in Talasaz to incorporate the application of the Bonferroni approach as taught in Liu because Liu teaches that doing so would provide a statistically powerful and computationally efficient method for measuring the associations between somatic mutations.
Claim(s) 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Talasaz (U.S. Patent Application Publication No. 2018/0023125), as cited on the IDS dated 02/27/2024, and Murtaza (U.S. Patent Application Publication No. 2019/0292575), and Jee (Jee et al.; Nature Letter, Vol. 534, pages 693-708, June 2016)as applied to claims 1-7, 14, 15, 18, 22, & 24-26, and further in view of Ståhlberg (Ståhlberg et al.; Nucleic Acids Research, Vol. 44, pages 1-7, March 2016), as cited on the IDS dated 02/27/2024.
The teachings of Talasaz, Murtaza, and Jee with respect to claim 1 is discussed above.
Regarding claim 21, Talasaz fails to teach the simultaneous amplification of target regions comprising at least 10 mutations in the cfDNA.
Ståhlberg teaches a high-level multiplexing and next-generation sequencing library construction from cfDNA liquid biopsies, specifically the amplification of 1 up to 31 different genomic DNA sequences in a single reaction (at least 10 mutations in the cfDNA) (pg. 3 column 2 2nd full paragraph lines 1-5), for the detection of multiple rare variants in the cfDNA sample (pg. 1 abstract lines 1-27). In addition, Ståhlberg teaches that this method is cost-efficient, flexible, and simple (pg. 6 paragraph bridging column 1 & 2 lines 35-40 & 49-63).
Talasaz, Murtaza, Jee, and Ståhlberg are considered to be analogous to the claimed invention because they are all in the same field of detection of rare variants in cfDNA samples. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of detection of genetic aberrations in a cfDNA sample taught in Talasaz to incorporate the multiplex detection of at least 10 mutations in the cfDNA sample as taught in Ståhlberg because Ståhlberg teaches that doing so would allow for detection of multiple rare variants in the cfDNA sample in a cost-efficient, flexible, and simple.
Claim(s) 27 & 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Talasaz (U.S. Patent Application Publication No. 2018/0023125), as cited on the IDS dated 02/27/2024, and Murtaza (U.S. Patent Application Publication No. 2019/0292575), and Jee (Jee et al.; Nature Letter, Vol. 534, pages 693-708, June 2016) as applied to claims 1-7, 14, 15, 18, 22, & 24-26, and further in view of Apte (Apte & Daniel; Cold Spring Harbor Protocols, Vol. 4, pages 1-10, March 2009).
The teachings of Talasaz, Murtaza, and Jee with respect to claims 1 & 2 are discussed above.
Regarding claim 27, Talasaz fails to teach the first target-specific primer have a melting temperature (Tm) range of 64-74°C and the second target-specific primers have a melting temperature (Tm) range of 56-60°C, however, the melting temperature is prima facie obvious in view of the routine nature of reaction condition optimization as taught in Apte. As set forth in the MPEP 2144.05IIA:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
Apte teaches protocols for primer design for nested PCR with an outer primer pair (first target-specific primers) and an inner primer pair (second target-specific primers) wherein the specificity of the PCR strongly depends on the melting temperature (Tm) of the primers determined by an equation to allow for optimization of the Tm of the primers (pg. 1 3rd full paragraph lines 1-5; pg. 1 5th full paragraph lines 1-3). Apte also teaches that in nested PCR the melting temperature of the inner nested primer pair (second target-specific) is significantly lower than that of the outer primer pairs (first target-specific primers) (pg. 4 7th full paragraph lines 1-5).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of designing first and second target-specific primers in Talasaz and Murtaza to incorporate designing nested primer pairs wherein the Tm of the inner nested primer pair (second target-specific primers) is significantly lower than the Tm of the outer primer pair (first target-specific primer) as taught in Apte to obtain a Tm of the first target-specific primers in a range of 68-74°C and a Tm of the second target-specific primers in a range of 56-60°C due to the routine nature of reaction condition optimization.
Regarding claim 28, Talasaz fails to teach the first target-specific primer have a melting temperature (Tm) range of 64-74°C and the second target-specific primers have a melting temperature (Tm) range of 56-60°C, however, the melting temperature is prima facie obvious in view of the routine nature of reaction condition optimization as taught in Apte. As set forth in the MPEP 2144.05IIA:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.)…
Apte teaches protocols for primer design for nested PCR with an outer primer pair (first target-specific primers) and an inner primer pair (second target-specific primers) wherein the specificity of the PCR strongly depends on the melting temperature (Tm) of the primers determined by an equation to allow for optimization of the Tm of the primers (pg. 1 3rd full paragraph lines 1-5; pg. 1 5th full paragraph lines 1-3). Apte also teaches that in nested PCR the melting temperature of the inner nested primer pair (second target-specific) is significantly lower than that of the outer primer pairs (first target-specific primers) (pg. 4 7th full paragraph lines 1-5).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of designing first and second target-specific primers in Talasaz and Murtaza to incorporate designing nested primer pairs wherein the Tm of the inner nested primer pair (second target-specific primers) is significantly lower than the Tm of the outer primer pair (first target-specific primer) as taught in Apte to obtain a Tm of the first target-specific primers in a range of 68-74°C and a Tm of the second target-specific primers in a range of 56-60°C due to the routine nature of reaction condition optimization.
Response to Arguments
The response traverses the rejection. The response asserts that the present amendments and remarks clarify the key point of novelty, how sequencing reads are constructed into read families for error suppression. Specifically, the response asserts that independent claims 1 and 2 now expressly require that each RF is formed using target sequence and fragment size and includes having different UMI sequences (i.e., an RF is not limited to reads sharing one UMI). Further, the response asserts that claim 1 recites that each grouped RF consists of reads including at least one read having a different UMI sequence and claim 2 similarly requires that a true mutation must be detected in all reads for at least two independent RFs. Further, the response assets that Talasaz attaches a barcode/tag to a parent polynucleotide prior to amplification and groups sequencing reads into families based on that tag such that each family corresponds to amplification products from a single tagged parent molecules (and thus has a single barcode/tag associated with that parent) and that Talasaz does not teach merging different barcode/tag groups into a singe RF and still treats reads having different barcodes/tags as corresponding to different parent molecules. Further, the response asserts that Talasaz’s copy-number variation discussion likewise does not cure the above deficiency as comparing sequences between sets of parent polynucleotides is different from applicants’ error suppression rule that a true mutation must be present in all reads across at least two independent RFs. These arguments have been thoroughly reviewed but were not found persuasive. First, the combination of Talasaz, Murtaza, and Jee, as applied to amended independent claims 1 & 2, as necessitated by amendment, appreciate a RF that is formed using target sequence and fragment size and includes having different UMI sequences. Specifically, as discussed further above, Jee teaches a method of attaching a barcode to target genomic DNA, linearly amplifying, attaching a second barcode, exponentially amplifying, sequencing, and then grouping the reads by both the primary barcode (first barcode corresponding to a read family) and the secondary barcode corresponding to a subfamily of reads (secondary different barcode corresponding to a read subfamily) with the same parent from a particular linear amplification step (each grouped read family consists of reads of one or more reads with a different UMI sequence) (pg. 697 column 1 2nd full paragraph lines 1-31; Extended Data Figure 2). Jee also teaches that the method of attaching and grouping read families with two different barcodes enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis (pg. 697 column 1 2nd full paragraph lines 15-21). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of grouping sequence reads of unique identity based on sequence information at the beginning (start) and end (stop) regions of the sequence reads, the length of the sequence read (fragment size), and the attachment of a barcode (UMI) as taught in Talasaz to incorporate the grouping the reads by both a primary barcode and the secondary barcode corresponding to a subfamily of reads (each grouped read family consists of reads of one or more reads with a different UMI sequence) as taught in Jee because Jee teaches that doing so would enable further reduction of the error rate by ensuring multiple reads from the linear amplification step are used in the analysis. Second, arguments presented cannot take place of evidence in the record. Further, Talasaz teaches determining consensus sequences for each family and that detecting copy number variation (genetic aberration) of consensus sequences between at least two sets of parent polynucleotides (a true mutation requires that the mutation is detected in all sequencing reads for at least two independent read families) (paragraph [0100] lines 4-8; paragraph [0124] lines 1-10; pg. 34 column 1 claim 1 lines 1-26).
The response also asserts that Murtaza does not teach (i) constructing a RF that intentionally spans multiple different UMI sequences using fragment length and target sequence, or (ii) requiring a mutation support across at least two independent RFs as criterion for a true mutation. These arguments have been thoroughly reviewed but were not found persuasive for the reasons set forth above.
The response also asserts that Liu is relied upon as allegedly teaching assessing associations between somatic mutations and gene expression in 12 cancer types and analysis with the Bonferroni approach, Ståhlberg is relied upon only as allegedly teaching multiplexing and next-generation sequencing library construction specifically fir the amplification of 1 up to 31 different sequences, and Apte is relied upon only as allegedly teaching protocols for primer design for nested PCR. Further, the response asserts that for each of these reasons, independent claims 1 and 2 are patentable over the cited references, whether those documents are considered alone or in combination with each other. Further, the response asserts that likewise, each dependent claims 3-7, 14, 15, 17, 18, 21, 22, and 24-28 depends from one of the independent claims either directly or indirectly and is submitted to be patentable for reasons similar to those discussed previously with regard to independent claims 1 and 2 , as well as for the additional claim elements each recites. These arguments have been thoroughly reviewed but were not found persuasive for the reasons set forth above.
For these reasons, and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Conclusion
Claims 1-7, 14, 15, 17, 18, 21, 22, & 24-28 are rejected.
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/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682
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