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
Status of the Claims
Claims 1-4, 6-9, 14-19, 21, 23-24, 27-28 and 34 are pending. Claims 1-4, 6, 8, 9 and 14-15 are the subject of this FINAL Office Action.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-4, 6-9, 14-15) and the oligo-primer configuration species of Figure 1 in the reply filed on 10/16/2025 is acknowledged. The elections read on claims 1-4, 6, 8, 9 and 14-15.
Claims 5, 7, 16-19, 21, 23-24, 27-28 and 34 are withdrawn.
Claim Rejections - 35 USC § 103- Maintained
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.
Claim(s) 1-4, 6, 8, 9 and 14-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over WOOD (US20210180121), in view of BIGLIA (US20160076089) as evidenced by US20140017685 and US20170233821.
The prior art as a whole demonstrates that it would have been obvious to a skilled artisan at the time of filing to detect SNPs using known high-sensitivity assays such as competitive blocker amplifications to achieve greater detection sensitivity in sequencing assays with a reasonable expectation of success.
As to claim 1, WOOD teaches a method for labeling and amplifying each strand of between 1 and 10,000 target genomic regions with an oligonucleotide barcode sequence by polymerase chain reaction (PCR), the method comprising:
(a) introducing into a DNA sample comprising the between 1 and 10,000 target genomic regions, for each target genomic region (Fig. 5):
(i) a first oligonucleotide F1/F2, comprising from 5′ to 3′ end, (A) a first region, (B) a second region with a length between 0 and 50 nucleotides, and (C) a third region targeting a first specific genomic region (Fig. 5, PCR I); and
(ii) a second oligonucleotide R1/R2, comprising from 5′ to 3′ end, (A) a fourth region, (B) a fifth region with a length between 0 and 50 nucleotides, (C) a sixth region comprising a unique molecular identifier (UMI) comprising at least four degenerate nucleotides, and (D) a seventh region targeting a second specific genomic region (Fig. 5, PCR I);
(b) performing at least two cycles of PCR amplification to generate a first PCR amplification product (2-3 cycles for first PCR in Fig. 5; paras. 0072-77);
(c) introducing into the first PCR amplification product:
(i) a third oligonucleotide A fwd comprising the first region (Fig. 5, PCR II); and
(ii) a fourth oligonucleotide A rev, comprising the fourth region (Fig. 5, PCR II);
(d) performing at least two cycles of PCR amplification, to generate a second PCR amplification product (2-4 cycles for second PCR in Fig. 5; paras. 0072-77));
(e) introducing to the second PCR amplification product:
(iii) a seventh oligonucleotide BT/A2A, comprising the fourth region (Fig. 5, PCR III); and
(f) performing at least two cycles of PCR amplification to generate a third PCR amplification product (multiple cycle in third PCR; paras. 0072-77).
As to claim 2, WOOD teaches the first region in the first oligonucleotide in step (a) and the fourth region in the second oligonucleotide in step (a) generate binding sites for universal amplification performed in step (c) (Fig. 5).
As to claim 3, WOOD teaches the fourth region in the second oligonucleotide comprises at least part of the next-generation sequencing (NGS) adapter sequence (Fig. 5).
As to claim 4, WOOD teaches the melting temperatures of the first and the fourth regions are between 0.01° C. and 10° C. higher than the melting temperatures of the third and the seventh regions (compare paras. 0051 & 0056 with para. 0039 (target-specific can be 56°C versus universal can be 72°C)).
As to claim 6, WOOD teaches the first PCR amplification product from step (d) is purified prior to step (e) using a method selected from the group consisting of SPRI purification, column purification, and enzymatic digestion (paras. 0031-32, 0068-69, 0072, 0079 & 0081).
As to claim 8, WOOD teaches (g) introducing to the PCR amplification product obtained in step (f), (i) an eighth oligonucleotide, comprising from 5′ to 3′ end, a ninth region and an eighth region, wherein the ninth region comprises at least part of the next-generation sequencing (NGS) adapter sequence, and optionally (ii) a ninth oligonucleotide, comprising the fourth region (Fig. 5); and
(h) performing at least one cycle of PCR amplification to obtain a third PCR amplification product (Fig. 5).
As to claim 9, WOOD teaches (i) adding NGS adapter sequences to the PCR amplification product obtained in step (f) by ligation reaction (ligation as a known option; para. 0003).
As to claim 14, WOOD teaches wherein an annealing temperature used in step (d) is between 0.01° C. and 10° C. higher than an annealing temperature used in step (b) (see claim 4; Examples, showing first anneal at 63°C and second at 64°C).
As to claim 15, WOOD teaches at least one of the between 1 and 10,000 target genomic regions is selected from the group consisting of is selected from the group consisting of AKT1, ALK, APC, AR, ATM, BRAF, CCND1, CDK4, CDKN2A, CHEK2, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FOXL2, GNA11, GNAQ, GNAS, HRAS, IDHL JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MLH1, MPL, MTOR, MYC, MYCN, MYD88, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RAF1, RB1, RET, ROS1, SF3B1, SMAD4, SMARCB1, SMO, STK11, and TP53 (NRAS, EGFR, KRAS, para. 0120).
WOOD does not teach (i) a fifth oligonucleotide (Blocker Displacement Amplification (BDA) forward primer) for each target genomic region, wherein the BDA forward primer comprises an eighth region targeting a specific genomic region, wherein the genomic region targeted by the eighth region is between 1 and 20 nucleotides closer to the seventh region compared to the genomic region targeted by the third region, (ii) a sixth oligonucleotide (BDA blocker) for each target genomic region, wherein 4 or more nucleotides at the 3′ end of the BDA forward primer sequence are also present at or near the 5′ end of the BDA blocker sequence; and wherein the BDA blocker contains a 3′ sequence or modification that prevents extension by a DNA polymerase, and wherein the concentration of the BDA blocker is at least 2 times that of the BDA forward primer. This, in summary, is a form of competitive blocker allele-specific PCR (CB-ASP), as explained in the specification.
However, CB-ASP, including the same assay claimed, has been routine in the art for decades, regularly used to achieve greater SNP-detection sensitivity. For example, BIGLIA teaches CB-ASP that uses a 3’-blocked oligo to competitively hybridize with a primer, the 3’-end of the primer overlapping with the 5’-end of the 3’-blocked oligo (Fig. 1). In fact, “the blocking oligonucleotide is added at a concentration that is at least twice, preferably at least three times, at least 4 times or at least 5 times higher than the concentration of the primer oligonucleotide it competes with” (BIGLIA, para. 0049). Further, “[t]he overlap (i.e. the common sequence between the competitor primer and the blocking oligonucleotide) can comprise, or consist of, at least 30%, 40%, 50%, 60%, 70%, 80% or 85% of the blocking oligonucleotide”; “[o]r the overlap can comprise, or consist of, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides” (para. 0047). Finally, “[t]he amplification of the target sequence can be followed by analysis of the amplified sequence, e.g. by a method to precisely determine the mutation(s) of the target sequence” such that “[o]nce the amplification or enrichment of the target sequence is complete, the sample may thus be further processed, e.g., subjected to a sequencing reaction” such as “Single-molecule sequencing, second generation high throughput sequencing, [and] pyrosequencing . . .” (paras. 0074-75). All of this increases sensitivity, as is known for decades (Example, 2. Sensitivity Test Results Using Oligo Blocks According to the Invention; see also US20140017685, para. 0099 (“One important factor affecting the sensitivity of detecting mutations which are present at a low frequency in a sample is the concentration ratio between the first primer and the blocking oligonucleotide probe. Generally, the concentration ratio of first primer/labelled oligonucleotide probe is less than one”) and US20170233821, Abstract & para. 0028 (“An ultra-sensitive, specific methodology for detecting PIK3CA mutations . . . presence of corresponding competitive blocking unlabeled probes for each exon can avoid non-specific amplification of wild-type PIK3CA sequence increasing the sensitivity and the specificity of method”; “This unlabeled blocking probe is used for competitive blocking of the wild type allele and is added at a higher concentration than the mutant allele specific primer, e.g. 5 to 20 times or 10 times higher concentration of the allele specific primer. . . . Thus, the unlabeled blocking probe competes with the allele specific primer for increased sensitivity”)). In other words, a skilled artisan would have been familiar with the routine use of CB-ASP using higher-concentration competitive blocker to achieve greater SNP detection and amplification sensitivity and specificity.
In sum, the prior art demonstrates conclusively that a skilled artisan would have been motivated to apply higher-concentration CB-ASP blockers to the SNP sequencing detection of the prior art such as WOOD to similarly achieve greater sequencing sensitivity and specificity with a reasonable expectation of success.
Response to Arguments
The rejection is maintained because Applicants’ arguments rely on elements not found in the claims. Most important, the claims do not require a sequencing step, much less any analysis of the PCRs or resulting amplicons, even less any specific data quality. Even if they did, contrary to Applicants’ assertions that sequencing is not taught in BIGLIA, in fact it is (paras. 0003-04, 0022 & 0074-75 and claim 15). BIGLIA provides explicit motivation to apply their blocker amplification enrichment technique to sequencing applications that Applicants’ skilled artisan would have known to include UMI/barcode-base library sequencing.
Yet, all of Applicants’ arguments rely on such unclaimed features. For example, Applicants argue that “Biglia fails to teach or suggest the quantitative sequencing
framework recited in the claims” (Reply, pg. 9). No such “quantitative sequencing” is found in the claims. In fact, claims 1 says nothing about sequencing. Instead, it is directed to “[a] method for labeling and amplifying each strand of between 1 and
10,000 target genomic regions with an oligonucleotide barcode sequence by polymerase chain reaction (PCR).” In other words, claim 1 is a PCR claim.
Applicants argue that there is a “technical incompatibility between the two approaches” of WOOD and BIGLIA because “Wood's use of UMIs is predicated on
maintaining a faithful correspondence between input molecules and sequencing reads, whereas Biglia's blocking strategy deliberately distorts that relationship to preferentially amplify variant sequences” (Reply, pg. 9). However, Applicants’ reasoning does not control the reasons to combine. The Examiner asserted that a skilled artisan desiring to target low-abundance mutations for amplification would be interested in such a blocking approach. There is nothing in WOOD that states what Applicants argue. Nothing in WOOD requires “maintaining a faithful correspondence between input molecules and sequencing reads.” Nor does anything in the claims. In fact, WOOD states that their disclosed method “enables overlapping primer pairs in a single tube for contiguous coverage over target regions, while simultaneously preventing amplification of both primer dimers and undesirable mini-amplicons that result from overlapping primer pairs” (Abstract). In other words, the method of WOOD is directed to, among other things, using UMIs to track multiple overlapping regions of a target region to generate contiguous coverage. If a skilled artisan was interested in this approach (contiguous coverage), then this also includes rare (e.g. 1% or less) events such as some SNPs. BIGLIA provides a common way to amplify low-abundance SNPs.
Applicants also argue that the claims require “propagation of those identifiers
through amplification,” however, in fact, nothing in the claims require this. Moreover, nothing in BIGLIA or WOOD indicates that blocker displacement would somehow not propagate the UMIs. On the contrary, BIGLIA states that “[t]here has been particular interest in the innovation of PCR stages that enable nondestructive selection and enrichment of mutant alleles, as this can improve sensitivity and credibility of downstream assays, such as standard sequencing analysis” (para. 0004; emphasis added). Thus, all mentions of sequencing with the blocker enrichment in BIGLIA presume what Applicants argue is missing.
Applicants argue that “[t]he claimed sequence enables quantitative analysis following enrichment by preserving molecule-level identity prior to introducing amplification bias” (Reply, pg. 10). Again, nothing in the claims requires this. Yet, it is not clear what Applicants’ mean by “prior to introducing amplification bias.” This possibly means the blocker enrichment step. However, it is unclear what in the claim preserves molecule-level identity before blocker enrichment. WOOD teaches the same steps before blocker enrichment. It seems WOODs results are no different as far as “preserving molecule-level identity prior to introducing amplification bias.” And as explained above, BIGLIA assumes as much in their technique (para. 0004).
Contrary to Applicants’ assertion, the Examiner did not “treat[] all overlapping primer/blocker systems as equivalent” (Reply, pg. 11). In fact, Applicants fail to explain where the Examiner did this. Regardless, the Examiner made clear that within the context of Applicants’ generic, broad claims, the prior art is replete with examples of blocker enrichment of step (e), all of which are known to enrich for rare SNP events for downstream detection, including sequencing. Then the Examiner provided additional references to support this analysis. Applicants fail to overcome this evidence.
Applicants assert, without evidence, that “UMI-based methods are known to substantially increase sequencing burden, often requiring an order-of-magnitude increase in sequencing reads and associated cost, while also introducing additional experimental complexity and bioinformatic processing requirements”; whereas, “blocker-based amplification methods, such as those described in Biglia, are designed to reduce sequencing requirements by enriching low-frequency variants prior to sequencing”; thus, “[a] person of ordinary skill in the art would therefore not have been motivated to incorporate UMis into a blocker-based amplification method, as doing so would undermine the efficiency advantages of enrichment while introducing additional cost and complexity, and the cited art provides no suggestion that such a combination would be beneficial” (Reply, pg. 11). Applicants improperly treat all UMI and all blocker-based amplification methods as equivalent. Various approaches simultaneously have burdens/costs and benefits, and a skilled artisan can easily weigh these based on the Application. If this was the standard for obviousness, then nothing would be obvious.
Finally, Applicants argue that none of the cited prior art teaches the third specific genomic region is positioned between the first and second specific genomic regions and is located 1 to 20 nucleotides closer to the second specific genomic region than the first specific genomic region. However, all that this requires is that some portion (e.g. one nucleotide) of the third specific genomic region (of any length) is closer to the reverse complement of the second specific genomic region than the first specific genomic region. The claim does not specify how this is measured (e.g. from 3’ or 5’ end of third specific genomic region, middle nucleotide, etc.). And 1 nucleotide closer in a 100bp region, for example, just means at base 51. In other words, the fifth oligo can have any nucleotide at base 51. Another way to put this is that the amplicon generated here is small/short. BIGLIA specifically states that “[t]he amplicon size is typically between about 60 to about 500 bp, preferably about 80 to about 250 bp” (para. 0036, for example). A small amplicon for the blocker amplification enrichment combined with a longer amplicon from the UMI reaction would yield the blocker oligo 1-20 bases closer to second specific genomic region than the first specific genomic region. Regardless, Applicants fail to present any data to support the criticality of this range.
Applicants should also beware that their arguments that the prior art is not enabling for their (unclaimed) invention does not snap back to provide grounds for non-enablement of their own invention. For example, if Applicants insist that WOOD and BIGLIA, which teach the same generic techniques here, are “technically incompatible,” then why isn’t Applicants’ invention also not enabled for the same reasons? Nothing here presents any specific unknown technique; so, what is it here that renders the invention enabled? How did Applicants not face the same supposed incompatibility (AKA non-enablement) for blocker enrichment applied to familiar barcoding schemes? As another example, Applicants argue that “Biglia's blocking strategy deliberately distorts that relationship to preferentially amplify variant sequences.” Doesn’t Applicants’ invention, using the same methods, do the same? Yet, Applicants contradict this statement when it comes to their own generic using of blocker enrichment: “[t]he claimed sequence enables quantitative analysis following enrichment by preserving molecule-level identity prior to introducing amplification bias.” In other words, Applicants’ arguments seem to be inconsistent when applied to their own invention versus the prior art.
Double Patenting- Obvious Type - Maintained
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory obviousness-type double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); and In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on a nonstatutory double patenting ground provided the conflicting application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement.
Effective January 1, 1994, a registered attorney or agent of record may sign a terminal disclaimer. A terminal disclaimer signed by the assignee must fully comply with 37 CFR 3.73(b).
Instant claims 1-4, 6, 8, 9 and 14-15 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1, 3-5, 9, 17, 22, 24-27, 29, 34-36, 40-41, 43, 47 and 52 of 17/420476, in view of BIGLIA (US20160076089) as evidenced by US20140017685 and US20170233821.
The instant claims are obvious over the conflicting claims because the conflicting claims teach the same three-PCR sequencing library preparation as the conflicting claims, and adding a CB-ASP reaction was a familiar option with familiar results. More specifically, the conflicting claims teach:
1. A method for preparing targeted regions of genomic DNA for high-throughput sequencing, the method comprising:
(a) obtaining a genomic DNA sample;
(b) amplifying at least a portion of the genomic DNA sample by performing two cycles of PCR using:
(i) a first oligonucleotide comprising, from 5′ to 3′, a first region, a second region having a length between 0 and 50 nucleotides, a third region comprising at least four degenerate nucleotides, and a fourth region comprising a sequence that is complementary to a first target genomic DNA region; and
(ii) a second oligonucleotide comprising, from 5′ to 3′, a fifth region, a sixth region having a length between 0 and 50 nucleotides, and a seventh region comprising a sequence that is complementary to a second target genomic DNA region;
(c) amplifying a product of step (b) by performing at least three cycles of PCR with an annealing temperature that is 0-10° C. higher than an annealing temperature used in step (b) and using:
(i) a third oligonucleotide comprising a sequence that is able to hybridize to the reverse complement of at least a portion of the first region; and
(ii) a fourth oligonucleotide comprising a sequence that is able to hybridize to the reverse complement of at least a portion of the fifth region; and
(d) amplifying a product of step (c) by performing at least one cycle of PCR using a fifth oligonucleotide comprising, from 5′ to 3′, an eighth region, a ninth region having a length between 0 and 50 nucleotides, and a tenth region comprising a sequence that is complementary to a third target genomic DNA region, wherein the third target genomic DNA region is at least one nucleotide closer to the first target genomic DNA region than the second target genomic DNA region;
wherein the resulting amplicons are suitable for quantitative next-generation sequencing to enable copy number variation (CNV) and allele ratio quantitation.
The conflicting claims do not explicitly teach (i) a fifth oligonucleotide (Blocker Displacement Amplification (BDA) forward primer) for each target genomic region, wherein the BDA forward primer comprises an eighth region targeting a specific genomic region, wherein the genomic region targeted by the eighth region is between 1 and 20 nucleotides closer to the seventh region compared to the genomic region targeted by the third region, (ii) a sixth oligonucleotide (BDA blocker) for each target genomic region, wherein 4 or more nucleotides at the 3′ end of the BDA forward primer sequence are also present at or near the 5′ end of the BDA blocker sequence; and wherein the BDA blocker contains a 3′ sequence or modification that prevents extension by a DNA polymerase, and wherein the concentration of the BDA blocker is at least 2 times that of the BDA forward primer. This, in summary, is a form of competitive blocker allele-specific PCR (CB-ASP), as explained in the specification.
However, CB-ASP, including the same assay claimed, has been routine in the art for decades, regularly used to achieve greater SNP-detection sensitivity. For example, BIGLIA teaches CB-ASP that uses a 3’-blocked oligo to competitively hybridize with a primer, the 3’-end of the primer overlapping with the 5’-end of the 3’-blocked oligo (Fig. 1). In fact, “the blocking oligonucleotide is added at a concentration that is at least twice, preferably at least three times, at least 4 times or at least 5 times higher than the concentration of the primer oligonucleotide it competes with” (BIGLIA, para. 0049). Further, “[t]he overlap (i.e. the common sequence between the competitor primer and the blocking oligonucleotide) can comprise, or consist of, at least 30%, 40%, 50%, 60%, 70%, 80% or 85% of the blocking oligonucleotide”; “[o]r the overlap can comprise, or consist of, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides” (para. 0047). Finally, “[t]he amplification of the target sequence can be followed by analysis of the amplified sequence, e.g. by a method to precisely determine the mutation(s) of the target sequence” such that “[o]nce the amplification or enrichment of the target sequence is complete, the sample may thus be further processed, e.g., subjected to a sequencing reaction” such as “Single-molecule sequencing, second generation high throughput sequencing, [and] pyrosequencing . . .” (paras. 0074-75). All of this increases sensitivity, as is known for decades (Example, 2. Sensitivity Test Results Using Oligo Blocks According to the Invention; see also US20140017685, para. 0099 (“One important factor affecting the sensitivity of detecting mutations which are present at a low frequency in a sample is the concentration ratio between the first primer and the blocking oligonucleotide probe. Generally, the concentration ratio of first primer/labelled oligonucleotide probe is less than one”) and US20170233821, Abstract & para. 0028 (“An ultra-sensitive, specific methodology for detecting PIK3CA mutations . . . presence of corresponding competitive blocking unlabeled probes for each exon can avoid non-specific amplification of wild-type PIK3CA sequence increasing the sensitivity and the specificity of method”; “This unlabeled blocking probe is used for competitive blocking of the wild type allele and is added at a higher concentration than the mutant allele specific primer, e.g. 5 to 20 times or 10 times higher concentration of the allele specific primer. . . . Thus, the unlabeled blocking probe competes with the allele specific primer for increased sensitivity”)). In other words, a skilled artisan would have been familiar with the routine use of CB-ASP using higher-concentration competitive blocker to achieve greater SNP detection and amplification sensitivity and specificity.
In sum, the prior art demonstrates conclusively that a skilled artisan would have been motivated to apply higher-concentration CB-ASP blockers to the SNP sequencing detection of the conflicting claims to similarly achieve greater sequencing sensitivity and specificity with a reasonable expectation of success.
Thus, the conflicting claims in light of the familiar CB-ASP prior art render obvious the instant claims.
Instant claims 1-4, 6, 8, 9 and 14-15 are rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over conflicting claims 1-12 of US12331350, in view of WOOD (US20210180121).
The instant claims are obvious over the conflicting claims because the conflicting claims teach the same CB-ASP reaction for sequencing library preparation, and three-PCR sequencing library preparation of the conflicting claims was a familiar option with familiar results. More specifically, the conflicting claims teach:
1. A method for simultaneously amplifying and detecting allelic variants at at least ten genetic loci, the method comprising:
(a) mixing a sample comprising DNA with a DNA polymerase and a blocker displacement amplification (BDA) oligo set for each genetic locus, each BDA oligo set comprising (i) a BDA forward primer, (ii) a BDA blocker, and (iii) a BDA reverse primer, wherein at least four nucleotides at the 3′ end of each BDA forward primer sequence are also present at or near the 5′ end of its respective BDA blocker sequence, wherein each BDA blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase, and wherein the concentration of each BDA blocker is at least twice that of its respective BDA forward primer; and
(b) subjecting the mixture to at least four cycles of amplification, thereby producing amplicons;
(c) performing next-generation sequencing (NGS) of the amplicons.
2. The method of claim 1, wherein the DNA polymerase has 3′ to 5′ exonuclease activity.
3. The method of claim 2, wherein each BDA blocker has a 3′ modification that prevents 3′ to 5′ exonuclease activity.
4. The method of claim 1, wherein the concentration of each BDA reverse primer and/or each BDA forward primer is determined based on a reads analysis of a previous calibration NGS experiment, wherein the concentration of each BDA reverse primer and/or each BDA forward primer is increased relative to the concentration used for the previous calibration NGS experiment.
7. The method of claim 1, wherein the BDA oligo set comprises at least 10 BDA oligo sets, each BDA oligo set comprising (i) a BDA forward primer, (ii) a BDA blocker, and (iii) a BDA reverse primer, wherein at least four nucleotides at the 3′ end of each BDA forward primer sequence are also present at or near the 5′ end of its corresponding BDA blocker sequence, wherein each BDA blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase, and wherein the concentration of each BDA blocker is at least twice that of its corresponding BDA forward primer, wherein each BDA blocker is complementary to a genomic region bearing a single nucleotide polymorphism (SNP) in which the alternative allele has a population frequency of between 10% and 90%, and wherein each corresponding BDA forward primer is not complementary to the SNP locus.
The conflicting claims do not explicitly teach the three-PCR technique of the conflicting claims.
However, this same technique was already known in the art to yield “overlapping primer pairs in a single tube for contiguous coverage over target regions, while simultaneously preventing amplification of both primer dimers and undesirable mini-amplicons that result from overlapping primer pairs” (WOOD, Abstract). Specifically, as explained above, WOOD teaches a method for labeling and amplifying each strand of between 1 and 10,000 target genomic regions with an oligonucleotide barcode sequence by polymerase chain reaction (PCR), the method comprising:
(a) introducing into a DNA sample comprising the between 1 and 10,000 target genomic regions, for each target genomic region (Fig. 5):
(i) a first oligonucleotide F1/F2, comprising from 5′ to 3′ end, (A) a first region, (B) a second region with a length between 0 and 50 nucleotides, and (C) a third region targeting a first specific genomic region (Fig. 5, PCR I); and
(ii) a second oligonucleotide R1/R2, comprising from 5′ to 3′ end, (A) a fourth region, (B) a fifth region with a length between 0 and 50 nucleotides, (C) a sixth region comprising a unique molecular identifier (UMI) comprising at least four degenerate nucleotides, and (D) a seventh region targeting a second specific genomic region (Fig. 5, PCR I);
(b) performing at least two cycles of PCR amplification to generate a first PCR amplification product (2-3 cycles for first PCR in Fig. 5; paras. 0072-77);
(c) introducing into the first PCR amplification product:
(i) a third oligonucleotide A fwd comprising the first region (Fig. 5, PCR II); and
(ii) a fourth oligonucleotide A rev, comprising the fourth region (Fig. 5, PCR II);
(d) performing at least two cycles of PCR amplification, to generate a second PCR amplification product (2-4 cycles for second PCR in Fig. 5; paras. 0072-77));
(e) introducing to the second PCR amplification product:
(iii) a seventh oligonucleotide BT/A2A, comprising the fourth region (Fig. 5, PCR III); and
(f) performing at least two cycles of PCR amplification to generate a third PCR amplification product (multiple cycle in third PCR; paras. 0072-77)
In sum, the prior art demonstrates that a skilled artisan would have been motivated to apply the three-PCR SNP loci sequencing library preparation technique of the prior art to the sequencing library SNP loci preparation technique of the conflicting claims to “overlapping primer pairs in a single tube for contiguous coverage over target regions, while simultaneously preventing amplification of both primer dimers and undesirable mini-amplicons that result from overlapping primer pairs” with a reasonable expectation of success.
Thus, the conflicting claims in light of the familiar multi-PCR sequencing library preparation prior art render obvious the instant claims.
Response to Arguments
The rejection is maintained for the reasons provided above.
Prior Art
The following prior art demonstrates that SNP-loci sequencing library preparation using barcodes and multiple PCR steps was well-known before effective filing of this application: US20200407798.
The following prior art demonstrates that SNP-loci sequencing library preparation using barcodes and blocking amplification was well-known before effective filing of this application: US20200332355.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
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/AARON A PRIEST/Primary Examiner, Art Unit 1681
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