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 .
Claims 1-20 are pending. Claims 1-8 are being examined on the merits. Claims 9-20 are withdrawn as being drawn to a non-elected invention.
Response to Restriction Requirement
The Response to Restriction Requirement filed January 23, 2026 has been entered.
Election/Restrictions
Applicant's election with traverse of Group I (claims 1-8) in the reply filed on January 23, 2026 is acknowledged. The traversal is directed to the restriction between Groups I and II, and is on the ground(s), in part, that instant claim 1 requires a single multiplex amplification reaction to amplify expressed target BCR nucleic acid template molecules that represent the IgH V-J coding sequence, IgL lambda V-J coding sequence and the IgL kappa V-J coding sequence, and that this combination of genes expressed amplified in a single multiplex amplification provides a technical contribution over the art. Applicant also argues that this feature is shared by claim 9 (Remarks, p. 3). This is not found persuasive, at least, because performing a single multiplex amplification to amplify expressed target BCR nucleic acid template molecules that represent the recited coding sequences does not provide a technical contribution over the art. For example, Kim (Deep sequencing of B cell repertoire, BMB Rep., 52(9): 540-547, 2019) teaches sequencing a library comprising the entire BCR repertoire where the library is generated, in part, by multiplex amplification of transcripts with a universal priming site attached to at least one end of the molecule (p. 541, right col., para. 1).
Claims 9-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on January 23, 2026.
The requirement is still deemed proper and is therefore made FINAL.
The Restriction Requirement mailed December 1, 2025 also included an Election of Species requirement for the various species of primers. Applicant’s election of group (c) directed to the primers of Tables 3 and 4 is also acknowledged.
All of elected claims 1-8 read on the elected species, and consequently no claims are withdrawn as a result of the species election.
Information Disclosure Statement
The Information Disclosure Statements submitted May 15, 2023, September 13, 2023 and November 21, 2024 have been considered.
Claim Objections
Claims 1-8 are objected to because of the following informalities:
Claims must have status identifiers (see MPEP 714 (II)(C)(A)).
Appropriate correction is required.
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 1-8 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
In considering whether there is sufficient evidence to support a determination that a
disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue”, the factors set forth in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988) should be considered. These factors are:
The breadth of the claims,
The nature of the invention,
The state of the prior art,
The level of one of ordinary skill in the art,
The level of predictability in the art,
The amount of direction provided by the inventor,
The existence of working examples; and
The quantity of experimentation to make or use the invention based on the content of the disclosure.
Each of these will be discussed in turn below.
Nature of the Invention and the Breadth of the Claims
The invention of independent claim 1 is drawn to a method for amplification of the expressed BCR repertoire in a target sample, comprising, in part, performing a single multiplex amplification reaction using a plurality of primer pairs, thereby resulting in amplicon molecules representing the target BCR repertoire in the sample. The dependent claims further describe the primers themselves or downstream processing steps.
Further, it should be noted that the elected species of primers is the primers of Tables 3 and 4. Table 3 describes 314 primers (i.e., SEQ ID NOs: 597-910), while Table 4 describes 40 primers (i.e., SEQ ID NOs: 911-950). Thus, the elected species of claim 1 is directed to every possible combination of the 314 primers of Table 3 and the 40 primers of Table 4, resulting in 10,000+ possible primer pair combinations. Thus, the elected species of the instant invention relates to highly-multiplexed PCRs used for targeted sequencing (as opposed to, e.g., whole genome amplification/sequencing).
Level of One of Ordinary Skill in the Art
One of ordinary skill in the art would have the knowledge equivalent to a graduate-level degree in molecular biology.
The State and Level of Predictability in the Art
The prior art generally teaches BCR repertoire sequencing, including methods using multiplex PCR (e.g., Bashford-Rogers1, “Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods”, BMC Immunology, BioMed Central, 15: 29, 2014; e.g., see Figs. 1-3). However, the prior art does not teach a highly-multiplexed PCR using 10,000+ primer pairs.
Prior to Applicant’s effective filing date, the prior art demonstrates that large plexing of PCR required extensive optimization and experimentation of primers, which Applicants fail to provide. Numerous references explain that multiplex PCR is generally limited to approximately 20-plex reactions. Shen2 (MPprimer: a program for reliable multiplex PCR primer design, BMC Bioinformatics, volume 11, Article number: 143 (2010), 3/18/2010) makes clear that “[t]he key step in running a successful multiplex PCR reaction is to design an optimal primer set combination” (p. 1). Rachlin3 (Computational tradeoffs in multiplex PCR assay design for SNP genotyping, BMC Genomics. 2005; 6: 102. Published online 2005 Jul 25. doi: 10.1186/1471-2164-6-102) states that “the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield” (Abstract). Stiller4 (Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA, Genome Res. 2009 Oct; 19(10): 1843–1848, 10/2009) states that “multiplex PCR amplification is commonly believed to produce overwhelming amounts of primer dimers and other spurious side products without extensive optimizations,” and notes numerous failed efforts in direct sequencing (p. 1844). Elnifro5 (Multiplex PCR: optimization and application in diagnostic virology, Clin Microbiol Rev, 13(4): 559-570, 2000) states that “[e]mpirical testing and a trial-and-error approach may have to be used when testing several primer pairs, because there are no means to predict the performance characteristics of a selected primer pair even among those that satisfy the general parameters of primer design” (p. 560).
Even more, these references explain that other essential parameters for successful multiplex PCR yield the need for extensive experimentation, yet these techniques fail to yield anything more than 20-30-plex reactions. Elnifro describes numerous other PCR components (e.g., cycling, polymerase, Mg2+ amounts, temperatures, buffers, etc.) that must be optimized, and suggests nested PCR, but only describes up to 20-30-plex reactions (pp. 560-68). Elnifro also states that “[t]horough evaluation and validation of new multiplex PCR procedures is essential” (pg. 568).
Shen also states that they could achieve only 20-plexing (Abstract). Yet, even this required overcoming extensive hurdles.
Multiplex polymerase chain reaction (PCR), defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates through the use of multiple primer sets (PS, comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical [1, 2] and environmental microbiology studies [3]. The key step in running a successful multiplex PCR reaction is to design an optimal primer set combination (PSC, a group of PSs, PSCs for primer set combinations). It is well known that for conventional PCR, the optimal PS has the following standards or properties: 1) primer size: 18-30 bp; 2) product size: 100-500 bp; 3) melting temperature (Tm) of both forward and reverse primers: 58-65°C, with a temperature difference of less than 3°C; 4) GC content of primers: 40-60%; 5) ΔG (Gibbs free energy) of the last five resides of the primers at the 3' end: ≥ -9 kcal/mol; etc[4, 5]. However, there are several additional criteria which must be taken into account when considering multiplex PCR assay: 1) lack of primer dimerization between all of the primers; 2) similarity of the Tms of each primer; 3) primer specificity to avoid mis-priming; and 4) constraint of electrophoretic mobility of the amplicons in order to separate and purify the DNA fragments easily in agarose gel electrophoresis [6–9].
It has been proved by Nicodeme and Steyaert that determining minimum-set primers for multiplex PCR is an NP-complete problem [10]. Most of the current programs mainly focus on the issue of determining the minimum-set primers, such as MPP (greedy algorithm) [11], PDA-MS/UniQ (modified compact genetic algorithm) [12], G-PRIMER (greedy algorithm) [13], and Greene SCPrimer (greedy algorithm) [14]. There are also other programs available for the design of primers in specific contexts. For example, Primaclade [15] designs minimally degenerate primers for comparative studies of multiple species. Primique [16] designs PCR primers specific for each sequence in a gene family. PrimerStation [8] designs human-specific multiplex PCR primers by checking the entire human genome database. MuPlex [5, 6] utilizes a multi-node graph algorithm derived from a greedy algorithm to assign and partition single nucleotide polymorphisms (SNP) into multiplex-compatible tubes for SNP genotyping. However, with the exception of PrimerStation, few of the programs mentioned above analyze the primer specificity against the genomic or transcript DNA database. Additionally, only a few of these programs provided a simple BLAST [17] search against the database locally or the GenBank database to examine the specificity of PCR primers [6, 11]. For example, the only database used to check primer specificity in PrimerStation is the human genome database. Moreover, few of the primer design programs constrain the amplicon size to allow separation and purification of the DNA fragments in agarose gel electrophoresis when designing PSCs for multiplex PCR. Some programs simply define a fixed length (for example, 10 bp) as the minimum size difference between the amplicons. However, the relation between an amplicons' size and their electrophoretic mobility in agarose gel electrophoresis is absolutely nonlinear [18]. For example, it is easy to separate two DNA fragments of 100 bp and 150 bp in agarose gel (0.5%-2%) electrophoresis, but quite difficult to separate two amplicons of 1000 bp and 1050 bp in a similar gel. The electrophoretic mobility of the amplicons should be considered at the very beginning of primer design.
[ . . . ]
To examine the performance of MPprimer, we designed a PSC comprising 5 PSs (10 primers) to amplify five mouse genes (β-actin, B2m, Pgk1, GAPDH, Rpl13a) in one tube as a typical multiplex PCR reaction. The result of the real experiment was nearly the same as that predicted by the virtual agarose gel electrophoresis analysis (Figure 3 and Figure 4). The homepage of MPprimer describes the detailed information of the experiment including the templates sequences, primer sequences, and the PCR and electrophoretic conditions. As another example, we have successfully used MPprimer to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions (see homepage of MPprimer for details).
Although designing minimum-set primers can save cost and time by reducing primer synthesis demand [11–14], it is crucial to design specific PSs, especially at the genomic or transcript level, to perform multiplex PCR with high reliability [9]. MPP [11], PDA-MS/UniQ [12], G-PRIMER [13] and Greene SCPrimer [14] are mainly concerned with the problem of minimum-set primers. PrimerStation [8] designs specific multiplex PCR primers only by checking the entire human genome database, but this database is too limited. The MPprimer web application supports transcript level specificity evaluation for more than ten species, while the stand-alone version can support any DNA sequence database, even the large genomic DNA database. MuPlex [5, 6] and MPP [11] simply use BLAST [17] to check primer specificity, but this is insufficient. Moreover, several other conditions such as Tm were not considered [9, 22]. Primaclade [15] and Greene SCPrimer [14] are used for degenerate primer design, while Primique [16] focuses on designing specific PCR primers for each sequence in a gene family. However, none of these programs provide the function for predicting the electrophoretic mobility of the amplicons from multiplex PCR reaction [18]. MuPlex [5, 6] and MPprimer use a very similar algorithm to find PSC in a graph where nodes are PSs and edges connect compatible pairwise PSs for multiplex PCR. The difference between them is that MPprimer selects nodes which have a lower penalty (indicating higher quality [4]) rather than random ones. Therefore, MPprimer can find the optimal PSC without enumerating and sorting all the PSCs to find the optimal one. It should be noted that, as our graph-expanding model is based on the preselected candidate primer sets (MPprimer utilizes Primer3 to design 5 primer sets for each of the template sequences), the output PSCs are not global but only local optimal. In another aspect, the running time of MPprimer is incomparable to other programs, because the specificity examination by MFEprimer requires more time for sequence similarity analysis between the primer sequence and the genomic or complementary DNA database of the same species. Therefore, the MPprimer web application currently only supports transcript level specificity examination. However, the stand-alone version of MPprimer supports unlimited databases, such as the genomic DNA database, which mainly depend on the user's computing capability
(pgs. 1-2 & 5-6; emphasis added).
Rachlin is even more explicit as to highly multiplexed PCRs:
Background
Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield.
Results
This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criteria. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels.
Conclusion
The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter may produce other adverse effects. The resulting design performance tradeoffs define a benchmark that can serve as the basis for comparing competing multiplex PCR design optimization algorithms and can also provide general rules-of-thumb to experimentalists seeking to understand the performance limits of standard multiplex PCR.
[ . . . ]
Multiplex PCR has recently emerged as a core enabling technology for high-throughput SNP genotyping [14-16], and variations on the standard protocol are being actively explored and in some cases more widely commercialized. It is in this context of genotyping that we focus our discussion of multiplex PCR assay design. Thus we will typically refer to multiplexing SNPs (rather than primers) but our treatment is readily applicable to most other PCR applications. Genomic variations in the form of Single Nucleotide Polymorphisms (SNPs) and associated haplotypes continue to garner tremendous interest particularly in the context of pharmacogenomic initiatives aimed at understanding the connection between individual genetic traits, drug response, and disease susceptibility [17-21]. Broad adaptation of genotyping technologies in clinical settings will depend on their cost and inherent clinical value and may be significantly impacted by ethical and legal considerations. Recent technological developments in PCR-based genotyping based on primer extension with universal PCR primers [22] have demonstrated very high (~100-plex) multiplexing levels, although the use of common primers does introduce other issues including the greater potential for cross-contamination.
Multiplex PCR assay design is a multi-objective optimization problem involving intrinsic performance tradeoffs. The key objectives we consider in this paper include the number of SNPs per tube (multiplex level) and the percentage of SNPs assigned to full tubes (coverage). We further require that all resulting tubes achieve uniform levels of multiplexing with the idea that doing so facilitates automation in a high-throughput environment. While lower coverage may be acceptable in initial survey studies involving many (104-106) SNPs, achieving high (>95%) coverage becomes obviously more important when the focus of investigation has been narrowed to a relatively small (102-103) set of SNPs each of which is suspected of having some biological or pharmacological impact.
The question we address in this paper is whether there are fundamental limitations to our ability to design assays that achieve multiplexing levels of arbitrary size using standard multiplex PCR protocols. While multiplex PCR is an established technique, its usefulness as the basis for future high-throughput platforms depends critically on scalability. We introduce a new framework of "multi-node graphs" to model the multiplex PCR problem. We show that the problem of finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability that two primers are mutually compatible drops below a critical threshold. This probability depends on fundamental primer selection criteria typically selected to avoid the formation of primer dimers. For standard criteria, we can identify where such a transition occurs, and show that it is consistent with typical multiplexing levels. The precise location of this critical transition point will also depend on N, the number of available SNPs. For a given level of coverage, the level of achievable multiplex is proportional to log(N). We further quantify design performance tradeoffs using two best-fit tube assignment algorithms on human SNP data.
[ . . . ]
. . . By sampling from chromosome 21 of the human genome, the actual probability that two SNPs are compatible is approximately 0.299. Figure Figure 1 would suggest, therefore, that designing 10-plex assays from N = 1,200 SNPs is generally straightforward, but that increasing multiplex performance to 15- to 20-plex or beyond becomes extremely problematic. This appears to be consistent with current design practice though we emphasize that the location of the phase transition depends on both the total number of SNPs and the number of candidate primer pairs per SNP. . . .
[ . . . ]
Next we employed the fixed-assignment best-fit algorithm to generate coverage curves for target multiplexing levels M = 10, 20, 30 while varying numbers of SNPs. We considered SNP sets containing between 100 and 1200 SNPs. Figure Figure33 presents our results. With 200 SNPs, 80% coverage could be achieved with 10-plex assays, but this drops to 40% coverage using 20-plex assays. However, if we increase the number of SNPs to 1200, then for 20-plex assays, coverage increases from approximately 40% to 80%. This graph shows that regardless of the multiplexing level desired, coverage increases with the number of SNPs but with diminishing returns. More precisely, for fixed multiplexing level M, coverage is roughly proportional to log(N).
(Abstract, pgs. 2, 3 & 4; emphases added). Pages 7-10 describe the extensive optimization required just to achieve up to 20-plex reactions. For example, “Using the above primer selection criteria, we generated an average of 1555.8 +/- 1249.4 primer pair candidates per SNP” (pg. 7). Thus, Rachlin demonstrates highly multiplexed SNP PCRs (as in the Specification) require extensive experimentation for each assay.
In sum, the art teaches that each application requires extensive experimentation for specific primers, specific reactions conditions, specific targets and specific assays, and only consistently achieves around 10-20-plexing.
Even in 2022, highly multiplexed direct sequencing was still very difficult. Xie6 et al (Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), Nat Commun. 2022 Apr 11;13(1):1881. doi: 10.1038/s41467-022-29500-4) explains that
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable
(Abstract).
The advance of high throughput sequencing has uncovered a large number of biomedically relevant DNA sequences, from driver mutations in cancer to new bacterial/viral pathogen DNA sequences to microbiome metagenomic profiles that affect mental disorders on the gut-brain axis1,2,3,4. For discovery applications, “shotgun” whole genome sequencing (WGS) is the preferred approach to identify novel DNA sequences of interest5. However, the human genome comprises over 3 billion nucleotides, and despite the lowering costs of high-throughput sequencing, it is not practical today to perform WGS to high depths necessary for identification of subclonal mutations, such as somatic mutations in cancer. For routine detection of disease-relevant DNA variants in known genes of interest, targeted sequencing or direct qPCR approaches are typically used6,7. Of the two dominant methods today for target enrichment, multiplex PCR tends to have shorter workflows and require less DNA input than hybrid-capture probes8. However, multiplex PCR struggles to scale to large panels covering hundreds of genes, due to the nonlinear increase of primer dimer species that reduce NGS mapping rates and increase effective cost9.
Currently, multiplex PCR methods for NGS target enrichment (e.g., Ampliseq8) primarily rely on (1) enzymatic digestion of modified bases in primers10 and (2) DNA size selection to preferentially remove short amplicon species likely to be primer dimers. However, both steps are labor-intensive and cannot be applied universally to all multiplexed PCR reactions. In contrast, relatively little systematic work has been reported on computational approaches to minimizing the formation of primer dimers in the first place. To the best of our knowledge, existing multiplex primer design algorithm never exceeded 70 primer pairs in one tube11,12,13,14. This is mainly due to the high computational cost when the number of primers increases15. The development of a robust multiplex primer set design algorithm that produces highly multiplexed primer sets with minimal primer dimer formation could allow further scaling of multiplex PCR target enrichment to even larger NGS panels when combined with enzymatic and size selection methods. Alternatively, it can simplify the workflow of moderate size NGS and qPCR assays by removing the need for strict contamination control from open-tube steps.
There are two primary challenges in designing highly multiplexed PCR primer sets: First, for an N-plex PCR primer set comprising 2N primers, there are (2N2) possible simple primer dimer interactions. For N = 50, this corresponds to (1002) = 4950 times as many potential primer dimer bindings as for a single-plex PCR primer set. Second, there are typically M > 10 reasonable candidate choices for each primer when considering specific gene targets and amplicon length constraints, resulting in M2N possible N-plex primer sets. For M = 20 and N = 50, the number of possible primer sets is 20100 ≈ 1.3 × 10130, billions of times larger than the number of atoms in the universe. Thus, it is computationally intractable to evaluate all possible multiplex primer sets. Simultaneously, primer dimer formation emerges from the interactions of two or more primers in the primer set, so changing the sequence of a primer to mitigate one primer dimer interaction may result in the appearance of another more serious primer dimer. In the language of numerical optimization, multiplex primer design is high dimensional problem with a highly non-convex fitness landscape. Consequently, standard convex optimization algorithms (e.g., gradient descent) will not be effective.
(pg. 2). Even in 2022, Xie had to design a specific “algorithmic framework for
designing highly multiplex PCR primer sets” (pg. 2). Thus, Xie also demonstrates that at the time of filing of the instant application, a skilled artisan would have expected to engage in extensive optimization and experimentation to yield a highly-multiplexed direct sequencing technique.
Most important, experts in directed sequencing consistently stated, even until 2015, that highly-multiplexed PCR-based directed sequencing does not work, and should be avoided. In 2015, Peng7 (Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes, BMC Genomics, 16:589, 2015) explained the many reasons why multiplex PCR was not used in direct sequencing before 2015:
There are multiple ways to enrich a target region before NGS. The most commonly used approaches are 1) hybridization capture from sequencing libraries using target specific probes [5] and 2) PCR amplification directly from sample DNA using target specific primers [6]. Although requiring more effort in up front primer design and chemistry optimization, many people still employ PCR amplicon based enrichment because, in general, the PCR process is easier to handle, requires less overall time, is more specific in terms of target sequence enrichment and can easily accommodate much lower DNA input. With the advent of high multiplex PCR, now hundreds to thousands of amplicons can be simultaneously amplified in one reaction, making the coverage of very large regions convenient [7].
Existing target enrichment, library preparation, and sequencing steps all utilize DNA polymerase and amplification processes, which introduce substantial bias (non-uniform amplification) and artifacts (polymerase errors generating sequence changes not present in the original samples). PCR amplification bias significantly affects quantification accuracy, because final sequence read counts may not accurately represent the relative abundance of original DNA and RNA fragments. Polymerase artifacts generated during the PCR cycles will most likely result in many “false” sequence variants present at low fractions in final sequence reads. These low level “false” variants cause difficulty in identifying real somatic mutations present at very low fraction (e.g. less than 2 %) in the sample. The root cause of these problems is the inability to distinguish the initial sampling of different original molecules from the resampling of the same molecule by primers during the PCR process. Such problems are exacerbated when more PCR cycles are needed to deal with low input DNA or poor quality DNA. PCR amplicon based target enrichment is more prone to these problems than the hybridization capture based enrichment for the following reasons. Random shearing or tagmentation process before hybridization capture creates random and diversified fragment ends, which can be used as a unique identifier for each starting DNA molecule [8]. Such unique identifiers offer a limited ability to keep track of different starting molecules and to remove PCR duplicates and associated amplification artifacts. PCR amplicon based enrichment loses such ability because all starting molecules are enriched with the same sequence ends for a given target specific amplicon.
To mitigate the problems of PCR duplication and biased amplification in NGS analysis, researchers have reported the inclusion of known number of synthetic internal standard molecules to improve the accuracy of NGS quantification [9]. Other approaches involve the use of exogenous molecular barcodes (or molecular tags) [8, 10, 11]. This is not to be confused with sample barcodes commonly used in current NGS workflows. The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Although molecular barcoding cannot prevent PCR duplication from happening, it provides a nice solution to track duplicates and treat them differently for downstream analysis. By employing molecular barcodes, polymerase artifacts generated during PCR can be distinguished from sequence variants present in original molecules. This barcoding has the potential to increase the detection accuracy for mutations at 1 % fraction or lower by removing low level false positives [8, 12, 13]. The target quantification can also be better achieved by counting the number of unique molecular barcodes in the reads rather than counting the number of total reads, as total read counts are more likely skewed for targets by non-uniform amplification [10, 14, 15].
Several variations of molecular barcodes have been successfully applied in NGS applications. Molecular barcodes have been incorporated into the ligation adapters during the library construction step for genome sequencing [13] and transcriptome sequencing [15]. In another study, barcodes were incorporated into molecular inversion probes for targeted somatic mutation detection [12]. Barcodes can also be incorporated into target specific PCR primers (in the form of a short stretch of random bases) in PCR amplicon sequencing [8, 10], thereby eliminating significant shortcomings in amplicon sequencing as mentioned earlier. In this aspect, so far all reported cases have been related to the amplification of one or a few amplicons by primers containing molecular barcodes, such as the analysis of a viral gene in an HIV resistance study [16], the analysis of 16srRNA gene in a human gut microbiota study [17], and the analysis of IG heavy chain in immune repertoire profiling [18]. As a result, those analyses have all been restricted to only very small regions. Thus, it will be beneficial if molecular barcodes can also be applied in high multiplex PCR amplicon sequencing. In order to accomplish this, some technical hurdles need to be overcome, e.g. how to avoid barcode resampling and how to suppress primer dimers in high multiplex PCR conditions
(pgs. 1-2). Stated simply, although highly multiplexed PCR was desired, it did not work. Rather, the prior art demonstrates that large plexing of PCR required extensive optimization and experimentation. Thus, this factor weighs against enablement because the state of the art at the time of effective filing until recently demonstrates that there were innumerable unpredictable hurdles to achieve the claimed highly-multiplexed amplification, and the specification fails to disclose any details essential to make and use such a method.
In addition, generally, the level of predictability in the biotechnology arts is low. C.f. In re Kubin, 561 F.3d 1351 (Fed. Cir. 2009); Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348 (Fed. Cir. 2007). This finding is further evidenced by the state of the prior art as explained above. As explained above, the prior art demonstrates that the claimed technique of 10,000 plus-plexing (or more) was unattainable prior the instant invention.
Amount of Direction Provided by Inventor and Existence of Working Examples
The specification recites numerous embodiments of different primer combinations that can be used in the method (e.g., paras. 36-41), and provides general guidelines on the amount of input RNA, primer concentrations and amplicon lengths, as well as barcoding amplicons (e.g., paras. 44-46, 48-50, 58). In addition, the specification describes “multiplex amplification” in general, and states …
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However, the specification fails to provide any details for the claimed 10,000 plus-plexing amplification as to the polymerase used and its concentration, the buffer(s) used and their concentrations or amounts, the reaction pH, PCR reaction conditions, any PCR additive used, Mg2+ amounts used, dNTP concentrations used, or any other important reaction condition variables known to affect multiplex PCRs (e.g., paras. 163, 232). The examples also do not provide a description of such highly-plexed reactions (e.g., Example 1: describes several embodiments of primer sets comprising primers in Tables 1-9, but does not teach how many primers were in each set (para. 242); Example 4 describes several embodiments of primer sets which were selected from the primers in Tables 6 and 10-11, but doesn’t teach how many primers were in each set (paras. 259-260). Therefore, in light of the evidence that highly multiplexed amplification required extensive experimentation for each application, and the lack of such guidance in the specification, this factor weighs against enablement.
Quantity of Experimentation Needed to Make or Use the Invention Based on the Content of the Disclosure
The above factors make clear that a skilled artisan would be required to engage in extensive optimization that amounts to undue experimentation. Although “an extended period of experimentation may not be undue if the skilled artisan is given sufficient direction or guidance,” yet here, the specification and prior art provide little guidance. See In re Colianni, 561 F.2d 220, 224 (CCPA 1977). In fact, the specification and state of the art indicate that a skilled artisan would expect that accomplishing highly-multiplexed amplification would require extensive, painstaking experimentation. Here, the specification is devoid of any guidance as to how to accomplish the claimed invention. Thus, the claims are not enabled.
Claims 2-8 depend from claim 1, and consequently incorporate the lack of enablement issues of claim 1.
Appropriate correction is required.
Claims 1-8 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.
As noted above, independent claim 1 is drawn to a method for amplification of expression nucleic acid sequence of a BCR repertoire, comprising, in part, performing a single multiplex amplification reaction using a plurality of primer pairs, thereby resulting in amplicon molecules representing the target BCR repertoire in the sample. Further, the elected species of primers is the primers of Tables 3 and 4. Table 3 describes 314 primers (i.e., SEQ ID NOs: 597-910), while Table 4 describes 40 primers (i.e., SEQ ID NOs: 911-950). Thus, the elected species of claim 1 is directed to every possible combination of the 314 primers of Table 3 and the 40 primers of Table 4, resulting in 10,000+ possible primer pair combinations. Thus, the elected species of the instant invention relates to highly-multiplexed PCRs used for targeted sequencing
As also noted above in conjunction with the lack of enablement rejection, it was not known in the art at the time of filing how to perform such highly plexed amplification reactions, nor does the instant specification provide guidance on how to do so.
Thus, the claimed embodiments directed to the elected species of these highly plexed amplification reactions were not describe in the specification in such a way as to reasonably convey to the ordinary artisan that the inventor had possession of the claimed elected species embodiments at the time the application was filed. Consequently, the embodiments directed to the elected species lacks written description support.
Claims 2-8 depend from claim 1, and consequently incorporate the lack of written description support issues of claim 1.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-8 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Indefiniteness rejections
Claim 1 recites the limitation “amplicon molecules representing the target BCR
repertoire in the sample”, the meaning of which is unclear. Specifically, it is unclear what characteristic(s) of the target BCR repertoire is intended to be “represent[ed]” in the amplicon molecules. For example, if each nucleic acid sequence comprised within the target BCR repertoire has at least one corresponding amplicon produced, is that “representing the target BCR repertoire”, or do the individual amplicon molecules comprised within the collective amplicon molecules have to have the same, perhaps, relative abundance as the original individual nucleic target acid sequences comprised within the original collective target nucleic acid sequences? Since the ordinary artisan would not be able to determine the metes and bounds of the claim, it is indefinite.
Claims 2-8 depend from claim 1, and consequently incorporate the indefiniteness issues
of claim 1.
Claim 3 recites the limitation “(ii) near or about the center nucleotide of the primer”,
the meaning of which is unclear. Specifically, it is not clear what the distinction is between “near” and “about”. The specification does not define either term, and the ordinary artisan would interpret them to have the same meaning. Since the ordinary artisan would not be able to determine the metes and bounds of the claim, it is indefinite.
Regarding claim 3, a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 3 recites the broad recitation of “each of the … plurality of primers includes … cleavable groups”, and the claim also recites “preferably located … near or at the termini … or … near or about the center” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Further, description of examples and preferences is properly set forth in the specification, rather than a claim. Since the ordinary artisan would not be able to determine the metes and bounds of the claim, it is indefinite.
Claim 8 refers to the primers listed in “Table[s] [9 and 6; 1 and 2; 3 and 4; 5]”. Where
possible, claims are to be complete in themselves. Reference to a specific table is permitted only in exceptional circumstances when there is no other practical way to define the invention in words. MPEP 2173.05(s). Here, clearly the primers listed in Tables 1-6 and 9 can be recited in the claims, and thus it is not a necessity for Applicant to refer to the tables.
Lack of antecedent basis rejections
Claim 8 recites the limitation "the at least one set of (i) … (ii) … (iii) and (iv)" in l. 1. There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 8 depends, recites using “a set of” each of the recited groups of primers, but does not recite using more than one set of each group of primers.
Prior Art
The prior art generally teaches BCR repertoire sequencing, including methods using multiplex PCR (e.g., Bashford-Rogers8, “Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods”, BMC Immunology, BioMed Central, 15: 29, 2014; e.g., see Figs. 1-3). However, the prior art does not teach a highly-multiplexed PCR using 10,000+ primer pairs.
Conclusion
Claims 1-8 are being examined, and are rejected. Claims 1-8 are also objected to. No claims are allowed.
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/CAROLYN L GREENE/Primary Examiner, Art Unit 1681
1 Bashford-Rogers was cited in the Information Disclosure Statement submitted November 21, 2024.
2 Shen was cited in the Information Disclosure Statement submitted November 21, 2024.
3 Rachlin was cited in the Information Disclosure Statement submitted November 21, 2024.
4 Stiller was cited in the Information Disclosure Statement submitted November 21, 2024.
5 Elnifro was cited in the Information Disclosure Statement submitted November 21, 2024.
6 Xie was cited in the Information Disclosure Statement submitted November 21, 2024.
7 Peng was cited in the Information Disclosure Statement submitted November 21, 2024.
8 Bashford-Rogers was cited in the Information Disclosure Statement submitted November 21, 2024.