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 .
Status of Claims
Claims 1-4 and 6-21 are currently pending. Claims 1, 3, 12-14 and 21 have been amended by Applicants’ amendment filed 11-06-2025. No claims have been added or canceled by Applicants’ amendment filed 11-06-2025.
Applicant's election by original presentation of Group I, claims 1-20, directed to a method for constructing a DNA library from a biological sample; and the election of Species, with traverse with regard to Species (A) and (B) as follows:
Species (A): wherein the first strand of the first adaptor further comprises a first primer recognition sequence over a 5' end of the molecule-specific barcode sequence (instant claim 12); and
Species (B): wherein the immobilized portion comprises a first coupling partner, configured to be able to stably bind to a second coupling partner attached to the solid support (instant claim 16), in the reply filed on June 3, 2024 was previously acknowledged.
Claims 6-11 and 15-20 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on June 3, 2024.
The restriction requirement was deemed proper and was made FINAL.
The claims will be examined insofar as they read on the elected species.
A complete reply to the final rejection must include cancellation of nonelected claims or other appropriate action (37 CFR 1.144) See MPEP § 821.01.
Therefore, claims 1-4, 12-14 and 21 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed December 23, 2020 is a CIP of US Patent Application 15908190, filed February 28, 2018 (now abandoned), which is a CON of 35 U.S.C. 371 national stage filing of International Application No. PCT/US2018/016778, filed on February 4, 2018, which claims the benefit of US Provisional Patent Application No. 62/482,189, filed April 6, 2017.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed November 6, 2025 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or objection not specifically addressed below are herein withdrawn.
Claim Rejections - 35 USC § 103
The rejection of claims 1-4, 12-14 and 21 is withdrawn under 35 U.S.C. 103 as being unpatentable over Kazakov et al. (hereinafter “Kazakov”) (US Patent No.11014957, issued May 25, 2021; PCT filed December 20, 2016; effective filing date December 21, 2015; of record) in view of Diagenode (Diagenode, 2013, 1-5; of record) as evidenced by Johnson et al. (hereinafter “Johnson”) (Journal of Molecular Evolution, 2023, 91, 263-280; of record); and ThermoFisher (ThermoFisher Scientific, 2006, 1-8; of record).
The combined references of Kazakov and Diagenode do not teach the sequential steps of preparing, ligating an adapter, synthesizing a complementary strand, ligating a second adapter, and amplification.
In view of the withdrawn rejection, Applicant’s arguments are rendered moot.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1-4, 12-14 and 21 is maintained under 35 U.S.C 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claim 1 is indefinite for the recitation of the term “the method consisting of the following steps” such as recited in claim 1, line 4 because it is unclear whether the method, “consisting of” the steps as recited will produce the library as claimed. For instance, the as-filed Specification teaches that after single-stranded extension reaction, the double-stranded molecule may have a 3’ overhang that needs to be removed to ensure high efficiency for any subsequent treatments, such as the ligation with a second adaptor (paragraph [0164]) and, thus, the metes and bounds of the claim cannot be determined.
Claim 1 is indefinite for the recitation of the term “immediately” such as recited in claim 1, lines 18, 23 and 29 because the instant as-filed Specification and original claims do not teach that the steps are carried out “immediately” after a previous step. Moreover, the term “immediately” is a relative term that renders the claim indefinite. The term “immediately” is not defined by the claim, and the Specification does not provide a standard for ascertaining the requisite amount of time period as compared to some other value that qualifies as an “immediately”, such that one of ordinary skill in the art would not be reasonably appraised of the scope of the invention and, thus, the metes and bounds of the claim cannot be determined.
Claim 12 is indefinite for the recitation of the terms “the first primer” and “the first primer recognition sequence” such as recited in claim 12, lines 8 and 9. There is insufficient antecedent basis for the terms “the first primer” and “the first primer recognition sequence” in the claim because claim 12, lines 2 and 7 recite the terms “a primer” and “a primer recognition sequence”.
Claim 21 is indefinite for the recitation of the term “when used for sequencing exhibits an error rate of no more than 2.25 x 10-10” such as recited in claim 21, lines 3-4 because claim 21 depends from claim 1, wherein claim 1 does not recite downstream procedures or analyses such as sequencing; the determination of an error rate; and/or detecting sequencing errors and, thus, the metes and bounds of the claim cannot be determined.
Claims 2-4, 13 and 14 are indefinite insofar as they ultimately depend from instant claim 1.
Claim Rejections - 35 USC § 112(d)
The rejection of claim 21 is maintained under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 21 recites (in part): “and wherein the barcoded library, when used for sequencing, exhibits an error rate of no more than 2.25 x 10-10” in lines 3-4 because claim 21 depends from claim 1, wherein claim 1 does not recite that there are downstream procedures/analyses such as sequencing; the determination of an error rate; and/or detecting sequencing errors. Thus, claim 21 is an improper dependent claims for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The rejection of claims 1-4, 12-14 and 21 is maintained under 35 U.S.C. 103 as being unpatentable over So et al. (hereinafter “So”) (US Patent Application Publication 20160281154, published September 29, 2016; of record) in view of Brown et al. (hereinafter “Brown”) US Patent Application Publication 20170247689, published August 31, 2017; and WO2016040524, filed September 9, 2015; published March 17, 2016; of record) as evidenced by Meyer et al. (hereinafter “Meyer”) (Nucleic Acids Research, 2007, 36(1), 1-7).
Regarding claims 1, 12 and 21, So teaches a method of preparing a single-stranded DNA library, comprising: (a) denaturing a double-stranded DNA fragment into single-stranded DNA (ssDNA) fragments; (b) removing 5' phosphates from the ssDNA fragments; (c) ligating single stranded primer docking oligonucleotides (pdo's) to 3' ends of the ssDNA fragments, wherein the pdo's are conjugated to a capture moiety capable of binding to an immobilized capturing reagent; (d) hybridizing primers to the pdo's, wherein the primers comprise a sequence complementary to the adaptor oligonucleotide sequence and comprise a first adaptor sequence that is at least 70% identical to a support-bound oligonucleotide coupled to a sequencing platform (interpreted as an adaptor comprising a primer binding site); (e) extending the hybridized primers to create duplexes, wherein each duplex comprises an single-stranded fragment and an extended primer strand; and immobilizing the duplexes to the immobilized capturing reagent (interpreted as synthesizing a complementary strand); (f) denaturing the double-stranded extension product, wherein the denaturing results in release of the extended primer strands from the immobilized capturing reagent and retention of the ssDNA fragments on the immobilized capturing reagent; and (g) collecting the extended primer strands, wherein the extended primer strands comprise the ssDNA library, wherein the first adaptor sequence comprises a barcode sequence (interpreted as fragmenting DNA; denaturing; dephosphorylating the 5’ end; ligating a barcoded adaptor to the 3’ end; primer binding site; extending; adding a second barcoded primer; producing a dsDNA library; dissociation followed by dephosphorylation; annealing a primer; and performing an extension reaction, claims 1, 3, 12 and 21) (paragraphs [0076]; and [0080]). So teaches that the invention provides a method, comprising: (a) hybridizing a target-selective oligonucleotide (TSO) to a single-stranded DNA (ssDNA) fragment in an ssDNA library to create a hybridization product; and (b) amplifying the hybridization product to create a double-stranded extension product, wherein the TSO comprises (i) a sequence that is complementary to a single target region and (ii) a first single-stranded adaptor sequence located at a first end of the TSO but not to both ends of the TSO, and wherein the ssDNA fragment comprises a second single-stranded adaptor sequence but does not comprise the first single-stranded adaptor sequence, wherein the second single-stranded adaptor sequence is located at a first end of the ssDNA fragment but not at both ends of the ssDNA fragment; and the amplifying comprises linear amplification (interpreted as ssDNA fragments; ligating an adaptor; amplifying resulting in adding a second adaptor, primer extension, dsDNA product; annealing a primer; and performing an extension reaction, claims 1, 12 and 21) (paragraph [0073]). So teaches that the term "barcode sequence" as used herein, generally refers to a unique sequence of nucleotides that can encode information about an assay, wherein a barcode sequence can encode information relating to the identity of an interrogated allele, identity of a target polynucleotide or genomic locus, identity of a sample, a subject, or any combination thereof, such that the barcode sequence can be a portion of a primer, a reporter probe, or both; and the barcode sequence can be located at the 5'-end or 3'-end of an oligonucleotide, or in any region of the oligonucleotide (interpreted as a molecule-specific barcode; inherently enabling accurate molecular identification in DNA samples with an input quantity of no less than 100 pg, claim 1) (paragraph [0124], lines 1-10). So teaches that ligation of an adaptor to a 3' end of a DNA fragment can comprise formation of a bond between a 3' OH group of the fragment and a 5' phosphate of the adaptor, such that removal of 5' phosphates from DNA fragments can minimize aberrant ligation of two library members, wherein at least 95% of 5' phosphates are removed from DNA fragments in a sample; and/or substantially all phosphate groups are removed from DNA fragments in a sample (interpreted as an adaptor comprising a phosphate; and dephosphorylation of the 5’ end of the ssDNA fragment, claim 1) (paragraph [0167]). So teaches that ssDNA can be prepared from dsDNA fragments by any means in the art or as described herein, by denaturation into single strands including by heat denaturation, claims 1 and 3) (paragraph [0168]-[0169]). So teaches that the DNA sequencing technology can utilize a 454 sequencing platform (Roche) (e.g. as described in Margulies, M. et al. Nature 437:376-380 [2005]), wherein 454 sequencing generally involves two steps including: (1) DNA can be sheared into fragments, such that the fragments can be blunt-ended; and oligonucleotide adaptors can be ligated to the ends of the fragments, wherein the adaptors generally serve as primers for amplification and sequencing of the fragments; then (2) beads can be captured in wells, which can be pico-liter sized, such that pyrosequencing can be performed on each DNA fragment in parallel (interpreting NGS sequencing as ligating double-stranded adaptors after synthesizing a complementary strand, claim 1) (paragraphs [0145]), wherein 454 sequencing adaptors are double-stranded as evidenced by Meyer (pg. 2, Figure 1). So teaches that DNA sequencing technology can utilize a SOLiD technology (APPLIED BIOSYSTEMS), wherein the SOLiD platform generally utilizes a sequencing-by-ligation approach, wherein library preparation for use with a SOLiD platform generally comprises ligation of adaptors are attached to the 5' and 3' ends of the fragments to generate a fragment library (interpreting NGS sequencing as ligating double-stranded adaptors after synthesizing a complementary strand, claim 1) (paragraph [0146], lines 1- 8).
Regarding claim 2, So teaches that a barcode sequence can have a length of about 4 to about 36 nucleotides (interpreted as encompassing 2-16 nucleotides, claim 2) (paragraph [0124], lines 18-20).
Regarding claim 3, So teaches that the DNA sequencing technology can utilize a 454 sequencing platform (Roche) (e.g. as described in Margulies, M. et al. Nature 437:376-380 [2005]). 454 sequencing generally involves two steps comprising: (1) DNA can be sheared into fragments, wherein the fragments can be blunt-ended; and (2) oligonucleotide adaptors can be ligated to the ends of the fragments, wherein the adaptors generally serve as primers for amplification and sequencing of the fragments (interpreted as shearing, claim 3) (paragraph [0145], lines 1-8). So teaches that dsDNA can be fragmented by any means known in the art or as described herein including that dsDNA can be fragmented by mechanical shearing, by nebulization, or by sonication (interpreted as shearing, claim 3) (paragraph [0163]).
Regarding claim 4, So teaches that the dsDNA fragments can be less than 800 bp, less than 500 bp, less than 200 bp, or less than 100 bp (interpreted as encompassing 50-500 bp, claim 4) (paragraph [0165], lines 1-5).
Regarding claims 13 and 14, So teaches that the Tm of the tso/target duplex can be between 0-100o C, between 20-90o C, between 40-80o C, between 50-70o C, or between 55-65o C, wherein the tso generally is sufficiently long to prime the synthesis of extension products in the presence of a polymerase; and the exact length and composition of a tso can depend on many factors, including temperature of the annealing reaction, source and composition of the primer, and ratio of primer:probe concentration, wherein the tso can be, for example, 8-50, 10-40, or 12-24 nucleotides in length (interpreted as encompassing annealing a first primer to each ssDNA molecule ligated to the single-stranded adapter, claims 13 and 14) (paragraph [0198]). So teaches that the exact length and composition of a primer can depend on many factors, including temperature of the annealing reaction, source and composition of the primer, and ratio of primer:probe concentration (interpreted as encompassing annealing a first primer to each ssDNA molecule ligated to the single-stranded adapter, claims 13 and 14) (paragraph [0221]). So teaches that the simplest equation for determining the melting temperature of primers smaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)), wherein computer programs can also be used to design primers, including but not limited to Array Designer Software, Oligonucleotide Probe Sequence Design Software, etc., such that the melting or annealing temperature of each primer can be calculated using software programs such as Oligo Design (interpreted as adjusting the annealing temperature, claims 13 and 14) (paragraph [0239]). So teaches that the PCR process is carried out as an automated process wherein the reaction mixture comprising template DNA is cycled through a denaturing step, a reporter probe and primer annealing step, and a synthesis step, whereby cleavage and displacement occurs simultaneously with primer-dependent template extension, wherein the automated process can be carried out using a PCR thermal cycler (interpreted as adjusting the annealing temperature to set a rate or 1-3oC/min, claims 13 and 14) (paragraph [0246]).
It is noted that per MPEP 2144.05(II)(A): “[G]enerally, 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).” Also noting that the claims do not recite the identity of any specific ssDNA, adapters, and/or primers, such that it is within the ability of one of ordinary skill in the art at the time of the invention to select or design appropriate primers, and to determine the conditions for annealing and/or extension.
So does not specifically exemplify increasing an initial temperature of no more than 20oC at a rate of 1-3oC per minute (claim 14, in part).
Regarding claim 14 (in part), Brown teaches rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein, which enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin (Abstract). Brown teaches independently tagged library constituents resulting in a library that allows easy recognition and elimination of artefactual errors in library generation, facilitating substantially more accurate nucleic acid sequencing (paragraph [0005], lines 17-20). Brown teaches annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule; extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence; obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, etc., wherein contacting a first primer comprises annealing said first primer to a nucleic acid of said target nucleic acid sample (paragraph [0010], lines 10-17). Brown teaches generating a labeled nucleic acid library comprising the steps of contacting a denatured library template to a first oligo population, an extension mix comprising dNTP and biotin-labeled ddNTP, and a low-processivity thermostable DNA polymerase to form a first strand composition, incubating the first strand composition in a temperature gradient incubator such that said first strand composition is subjected to a temperature ramp from a first oligo population annealing temperature to a denaturing temperature (interpreted as raising the annealing temperature at a rate, claim 14) (paragraph [0025], lines 1-10). Brown teaches a method of generating tagged fragments of a nucleic acid sample comprises the steps of contacting the nucleic acid sample to an oligonucleotide library comprising an oligonucleotide having a sequence not identical to any sequence of the nucleic acid sample and a nucleic acid extension composition comprising dNTP, an affinity tag, and a DNA polymerase, to form affinity-tagged, oligo-tagged fragments of the nucleic acid sample and affinity purifying the affinity-tagged, oligo-tagged fragments of the nucleic acid sample (paragraph [0027], lines 3-12). Brown teaches that hybridization temperatures are at least about 2oC to about 6oC lower than the melting temperature (Tm) (paragraph [0067], last three lines). Brown teaches that the term "melting temperature" or "Tm" commonly refers to the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands, wherein equations for calculating the Tm of nucleic acids are well known in the art including one equation that gives a simple estimate of the Tm value is as follows: Tm=Sl.5+ 16.6(log 1 0[Na+])0.41 (¾[G+C])-675/n-l.0 m, when a nucleic acid is in aqueous solution having cation concentrations of 0.5 M or less, the (G+C) content is between 30% and 70%, n is the number of bases, and m is the percentage of base pair mismatches (interpreted as melting temperature can be 30-35oC, claim 14) (paragraph [0082]). Brown teaches that the mixture is heated to a temperature consistent with polymerase activity, such as for example, 20° C, 21° C, 22° C, 23° C., 24° C, 25° C, 26° C, 27° C, 28° C, 29° C. 30° C, 31° C, 32° C, 33° C, 34° C, 35° C, 36° C, 37° C, 38° C, 39° C., 40° C., 41° C., 42° C., or a number greater or less than a number in this range), and incubated for a period sufficient to synthesize the first strand library (interpreted as encompassing a starting temperature of 20oC, claim 14) (paragraph [0163]). Brown teaches that the thermal cycler performs a program comprising: (1) maintaining the temperature at about a low temperature for a period of time, (2) increasing the temperature to a DNA annealing temperature, (3) maintaining at the annealing temperature for a period of time, (4) increasing the temperature to a denature temperature for a period of time, repeating (1) to (4) for at least 9 times; or as an alternative, the thermal cycler can maintain the temperature at about 16° C for about 3 minutes, wherein the temperature from (1) to (2) is increased slowly, such that the temperature is ramped up by a small increment of temperature at about 0.1° C/second; such that the temperature of (2) is about 45° C., about 50° C, about 55° C, about 60° C, about 65° C, about 68° C, about 70° C, or more; and/or the temperature of (2) is slowly ramped up to about 60° C by 0.1° C/second; and that in some cases, the temperature of (2) is the same as the temperature of (3), and/or the temperature of (2) is further increased to reach the temperature of (3) (interpreted as encompassing a rate of 1-3oC/minute, claim 14) (paragraphs [0332]). Brown teaches that Figure 1 illustrates a schematic of the Rapid Library Prep utilizing genomic DNA as the target nucleic acid sample (interpreted as a DNA from a biological sample, claim 1) (paragraph [0032]; and Figure 1). Brown teaches the preparation of a library is performed in Figures 1A-1G, comprising:
Step 1 – a target nucleic acid sequence comprising genomic DNA is bound by multiple random oligonucleotide ("Random 8-mer") primers containing 5' sequencing adapter tails ("A-adapters") (FIG. 1A) (interpreted as ligating a single-stranded adaptor to a 3’ end of the ssDNA molecules, claim 1i);
Step 2 – a pool of nucleotides containing a ratio of deoxy-NTPs (dNTPs) to biotinylated-dideoxy NTPs (ddNTPs) and reaction buffer is added to this mixture; and a DNA polymerase having strand displacement activity and ddNTP/biotin incorporation ability is added and extension progresses from the 3' OH of the random oligonucleotides until a biotinylated-ddNTP ("Biotin ddNTP") is incorporated, at which point extension terminates, as shown in step 2 (FIG. 1B) (interpreted as attaching to a bead, claim 1ii);
Step 3 - streptavidin-coated magnetic beads are then added to isolate the tagged first strand extension product; and a second set of random oligonucleotide ("Random 8-mer") primers containing 5' sequencing adapter tails ("B adapters") is combined with the isolated first strand extension product, a pool of dNTPs, reaction buffer, and a DNA polymerase having strand displacement activity; and a complementary second strand is generated forming a double-stranded molecule as shown in step 3 (FIG. 1C) (interpreted as synthesizing a complementary strand, claim 1iii);
Step 4 - the double-stranded product is washed and the displaced product is removed as shown in step 4 (FIG. 1D and FIG. 1E), wherein the biotin tag can be removed at this step (interpreted as forming double-stranded linear DNA molecules, claim 1iv); and
Step 5 - full-length adapter sequences are added via PCR amplification as shown in step 5 (FIG. 1F), and the resulting molecule in FIG. 1G is suitable for sequencing via any of the sequencing methods described herein (interpreting adding full length adapter sequences as ligating a second double-stranded adapter; and immediately performing amplification, claim 1iv-v) (paragraph [0112]).
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It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of annealing an oligonucleotide to a nucleic acid molecule as exemplified by Brown, it would have been prima facie obvious before the effective filing date of the claimed invention to modify the method of preparing DNA sequencing libraries from a biological sample including cfDNA, the method comprising: dephosphorylating and denaturing dsDNA into ssDNA, fragmenting ssDNA, ligating a single-stranded barcoded adaptor to the 3’ end of each ssDNA fragment, synthesizing complementary strand, ligating a second adaptor to the dsDNA molecule including an Illumina-specific adaptor oligonucleotide; extending, and denaturing the dsDNA molecule into ssDNA extension products as exemplified by So to include the reagents and methods for the rapid preparation of nucleic acid libraries including ddNTPs, primers, tags, barcodes, enzymes, annealing temperatures, and/or slow temperature ramp rates such as within a thermocycler as disclosed by Brown, with a reasonable expectation of success in accurately generating DNA libraries from nucleic acid samples; in minimizing the formation of artifactual chimers; in improving the efficiency of adaptor ligation by at least 10-fold; in allowing for the independent verification of sequence information; and/or in the detection and/or measurement of mutations such as single nucleotide polymorphisms (SNPs) associated with a number of diseases including cancer.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly rejected under 35 USC §103 as obvious over the art.
Response to Arguments
Applicant’s remarks filed November 6, 2025 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) So and Brown do not teach that the second adaptor is a double-stranded adaptor and subsequently amplifying the double-stranded molecules to form a DNA library (Applicant Remarks, pg. 11, last full paragraph through pg. 12, first full paragraph).
Regarding (a), although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). Moreover, although none of the references has to teach each and every claim limitation. If they did, this would have been anticipation and not an obviousness-type rejection. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Moreover, as noted in MPEP 2112.01(I),
where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Applicant’s assertion that So and Brown do not teach that the second adaptor is a double-stranded adaptor and subsequently amplifying the double-stranded molecules to form a DNA library, is not found persuasive. The Examiner contends that the combined references of So and Brown teach all of the limitations of the claims. Although So teaches that an embodiment that includes the ligation of a second single-stranded adapter, So also teaches that the
So also teaches:
A first adaptor sequence at least 70% identical to a support-bound oligonucleotide coupled to a sequencing platform; and extending the primers to create duplexes (interpreted as synthesizing a complementary sequence; ligating double-stranded adaptors; and sequencing picoliter amounts the DNA molecules, claim 1) (paragraph [0076]).
Fragments attached to beads can be PCR amplified within droplets, resulting in amplified DNA fragments on each bead (interpreted as ligating a second double-stranded adaptor; synthesizing a complementary sequence); and amplifying dsDNA, claim 1) (paragraph [0076]).
Next Generation Sequencing platforms including Illumina, 454 Life Sciences, Helicos Biosciences, Solexa, Genome Analyzer, etc., wherein 454 sequencing adaptors are double-stranded as evidenced by Meyer (interpreting NGS sequencing as ligating a second double-stranded adaptor, claim 1) (paragraph [0139]-[0140]).
Brown teaches:
The preparation of a library is performed in Figures 1A-1G, comprising:
Step 1 – a target nucleic acid sequence comprising genomic DNA is bound by multiple random oligonucleotide ("Random 8-mer") primers containing 5' sequencing adapter tails ("A-adapters") (FIG. 1A) (interpreted as ligating a single-stranded adaptor to a 3’ end of the ssDNA molecules, claim 1i);
Step 2 – a pool of nucleotides containing a ratio of deoxy-NTPs (dNTPs) to biotinylated-dideoxy NTPs (ddNTPs) and reaction buffer is added to this mixture; and a DNA polymerase having strand displacement activity and ddNTP/biotin incorporation ability is added and extension progresses from the 3' OH of the random oligonucleotides until a biotinylated-ddNTP ("Biotin ddNTP") is incorporated, at which point extension terminates, as shown in step 2 (FIG. 1B) (interpreted as attaching to a bead, claim 1ii);
Step 3 - streptavidin-coated magnetic beads are then added to isolate the tagged first strand extension product; and a second set of random oligonucleotide ("Random 8-mer") primers containing 5' sequencing adapter tails ("B adapters") is combined with the isolated first strand extension product, a pool of dNTPs, reaction buffer, and a DNA polymerase having strand displacement activity; and a complementary second strand is generated forming a double-stranded molecule as shown in step 3 (FIG. 1C) (interpreted as synthesizing a complementary strand, claim 1iii);
Step 4 - the double-stranded product is washed and the displaced product is removed as shown in step 4 (FIG. 1D and FIG. 1E), wherein the biotin tag can be removed at this step (interpreted as forming double-stranded linear DNA molecules, claim 1iv); and
Step 5 - full-length adapter sequences are added via PCR amplification as shown in step 5 (FIG. 1F), and the resulting molecule in FIG. 1G is suitable for sequencing via any of the sequencing methods described herein (interpreting adding full length adapter sequences as ligating a second double-stranded adapter; and immediately performing amplification, claim 1iv-v).
The combined references of So and Brown teach all of the limitations of the claims including ligating second double-stranded adaptors to dsDNA molecules. Thus, the claims remain rejected.
New Objections/Rejections
Claim Rejections - 35 USC § 112(a) – New Matter
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 1-4, 12-14 and 21 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection. This is a new rejection necessitated by amendment of the claims in the response filed 11-06-2025.
MPEP § 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims” and “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112, para. 1, as lacking adequate written description”. According to MPEP § 2163.I.B, “While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure” and “The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117”.
The claim contains subject matter that was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art (hereafter the Artisan), that the inventor(s), at the time the application was filed, had possession of the claimed invention. 37 CFR §1.118 (a) states that "No amendment shall introduce new matter into the disclosure of an application after the filing date of the application". Claim 1 recites in part: “ligating a single-stranded adaptor to a 3’ end of each of the plurality of single-stranded DNA molecules” in lines 9-10; “immediately after the ligating the single-stranded adaptor, synthesizing a complementary strand” in lines 18-19; “immediately after the synthesizing the complementary strand, ligating a second adaptor” in lines 23-24; and “immediately after the ligating the second adaptor, performing amplification of the dsDNA molecules” in line 29. Applicant does not point to where support for the instant amendments can be found in the as-filed Specification, such that it is unclear where in the as-filed Specification this limitation is taught.
Upon review of the instant as-filed Specification and original claims, support was not found for the ligating single-stranded adaptors to the 3’ end of ssDNA molecules and/or for performing many of the reaction steps ‘immediately’ after a previous step. The instant as-filed Specification, filed December 23, 2020 teaches, for example; “preparing a DNA sample from a biological sample…synthesizing a complementary strand” (paragraphs [010]-[012]); “the method further comprises: immobilizing each of the plurality of ssDNA molecules ligated to the first strand of the first adaptor to the solid support” (paragraph [032]; “Between the ligating a second adaptor to a free end of the dsDNA molecule corresponding to the each of the plurality of ssDNA molecules immobilized to the solid support at an immobilized end thereof and the performing a PCR amplification” (paragraph [038]); and “in the kit, the first adaptor can be single-stranded” (paragraph [048]. No such corresponding teaching of using ligating a single-stranded adaptor to the 3’ end of ssDNA; and/or performing some steps ‘immediately’ after a previous step is taught in the instant as-filed Specification.
A claim-by-claim analysis and for dependent claim 1, and a method step by method step analysis regarding where support can be found for the these teachings in the originally filed specification is respectfully suggested. See MPEP § 2163 particularly § 2163.06.
Claims 1-4, 12-14 and 21 will remain rejected until Applicant cancels all new matter.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and
103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for
the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 12-14 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Gansauge et al. (hereinafter “Gansauge”) (Nucleic Acids Research, January 2017, 45(10), 1-10; and Supplementary Materials, 1-17) in view of Head et al. (hereinafter “Head”) (BioTechniques, 2014, 56(1), 61-77) as evidenced by Bashkirov et al. (hereinafter “Bashkirov”) (US Patent No. 10246736, issued April 2, 2019; effective filing date February 15, 2008); and Gansauge et al. (hereinafter Gansauge 2013) (Nature Protocols, 2013, 15, 2279-2300); and Kircher et al. (hereinafter “Kircher”) (Nucleic Acids Research, 2012, 40(1), 1-8). This is a new matter rejection. This is a new rejection necessitated by amendment of the claims in the response filed 11-06-2025.
Regarding claim 1, Gansauge teaches a ssDNA2.0 which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of 8 random bases (interpreted as comprising a molecules specific barcode; and barcode sequences of 2-16 nucleotides, claim 1) (Abstract, lines 8-10). Gansauge teaches ssDNA2.0 for single-stranded library preparation, which tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and more compatible with automation, and increasing library yields from tissues stored in formalin for many years by several orders of magnitude (interpreted as preparing linear DNA libraries, claim 1) (pg. 1, Abstract, lines 20-28). Gansauge teaches a graphical outline of a method is shown in Figure 1B, where
DNA fragments are dephosphorylated and denatured, after which the first adapter is joined to their 3’-ends using CircLigase, wherein ligated DNA strands are immobilized on streptavidin-coated magnetic beads, copying the template strand with a DNA polymerase, the generation of blunt ends and the ligation of the second adapter, are carried out on beads, thereby minimizing losses of DNA in intermittent purification steps (interpreted as preparing a DNA sample; ligating a single-stranded adaptor to a 3’ end; synthesizing a complementary strand; and ligating a second double-stranded adaptor, claim 1) (pg. 1, col 2, last partial paragraph; and Figure 1B). Figure 1B is shown below:
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Gansauge teaches that in DNA2.0 were prepared from 0 to 27 ml DNA extract, wherein CircLigase-based library preparation was performed as described previously, wherein libraries are amplified and indexed via PCR with 5’ tailed primers as evidenced by Gansauge 2013 (pg. 2881, Figure 1b) with the only major modification being a 3’-5’ exonuclease treatment of the single-stranded adapter oligonucleotide CL 78 using the Klenow fragment of Escherichia coli DNA polymerase I in the absence of nucleotides in order to remove synthesis artifacts and potential DNA contamination, such that ssDNA2.0 library preparation differed from this method in three aspects including: (1) single-stranded ligation of the first adapter oligonucleotide was carried out using T4 DNA ligase in the presence of a splinter oligonucleotide; (2) an additional 45oC wash step was introduced to remove the splinter oligonucleotides after immobilization of the ligation products on streptavidin-coated beads; and (3) copies of the template strands were created using an extension primer protected by phosphorothioate (PTO) linkages (CL130) in combination with Klenow fragment instead of Bst DNA polymerase, which avoided blunt-end repair and associated bead wash steps (steps 16–19 in Gansauge and Meyer as evidenced by Gansauge 2013) (pg. 4, first full paragraph). Gansauge teaches that double-stranded libraries were prepared using two methods: (1) following the method described in Meyer and Kircher for highly degraded DNA but omitting the final purification step after adapter fill-in to maximize recovery of library molecules; and (2) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (interpreted as forming linear double-stranded DNA libraries, claim 1) (pg. 5, col 1, first full paragraph). Gansauge teaches that all libraries were quantified by quantitative polymerase chain reaction (PCR) as described elsewhere, such that for comparisons of library yields, molecule counts obtained for the libraries prepared with the method of Meyer and Kircher (interpreted as the adaptor comprising a primer recognition sequence; and forming linear dsDNA molecules; and PCR as performing amplification, claim 1) (pg. 5, col 1, second full paragraph).
Regarding claim 2, Gansauge teaches a ssDNA2.0 which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of 8 random bases (interpreted as comprising a molecules specific barcode; and barcode sequences of 2-16 nucleotides, claim 2) (Abstract, lines 8-10). Gansauge teaches that in DNA2.0 were prepared from 0 to 27 ml DNA extract, wherein CircLigase-based library preparation was performed as described previously, wherein libraries are amplified and indexed via PCR with 5’ tailed primers as evidenced by Gansauge 2013 (pg. 2881, Figure 1b).
Regarding claims 3 and 4, Gansauge teaches that fragmented human genomic DNA was obtained by shearing 1 mg of human DNA using a Covaris S2 ultrasonicator (interpreted as shearing, claim 3) (pg. 4, col 1; first full paragraph, lines 1-3), where it is known that the Covaris S2 sonicator can fragment purified nucleic acids in the range of 60 to 90 base pair fragments as evidenced by Bashkirov (col 3, lines 17-22). Gansauge teaches that DNA fragments are 5’ and 3’ dephosphorylated and separated into single strands by heat denaturation (interpreted as dephosphorylation followed by dissociation, claim 3) (pg. 2, Figure 1A).
Regarding claim 12 (in part), Gansauge teaches that ssDNA2.0 library preparation differed from this method in three aspects including: (1) single-stranded ligation of the first adapter oligonucleotide was carried out using T4 DNA ligase in the presence of a splinter oligonucleotide; (2) an additional 45oC wash step was introduced to remove the splinter oligonucleotides after immobilization of the ligation products on streptavidin-coated beads; and (3) copies of the template strands were created using an extension primer protected by phosphorothioate (PTO) linkages (CL130) in combination with Klenow fragment instead of Bst DNA polymerase, which avoided blunt-end repair and associated bead wash steps (steps 16–19 in Gansauge and Meyer) (interpreted as a single stranded adaptor comprising primer recognition sequence; and synthesizing a complementary strand includes annealing a primer and performing an extension reaction, claim 12) (pg. 4, first full paragraph).
Regarding claims 13 and 14, Gansauge teaches that for single-stranded DNA ligation adapters CL78, TL128, TL103 or TL134 were used, and the mixture was heated up to 95oC for 10 s in a thermal cycler, followed by a ramp to 10oC at 0.1oC/s (interpreted as a starting temperature of 0oC, encompassing 20-25oC and raising the temp encompassing 30-35oC; and a rate of 1-3oC per minute, claims 13 and 14) (pg. 4, col 1, last partial paragraph). Gansauge teaches in Supplementary Materials a list of primers comprising PTO linkages and extension primers (Supplementary Materials, pg. 16, Supplementary Table 2). It is noted that per MPEP 2144.05(II)(A): “[G]enerally, 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).” Also noting that the claims do not recite the identity of any specific ssDNA, adapters, and/or primers, such that it is within the ability of one of ordinary skill in the art at the time of the invention to select or design appropriate primers, and to determine the conditions for annealing and/or extension
Regarding claim 21, Gansauge teaches that 10 microliters of each of single-stranded and double-stranded library were amplified and double-indexed using AccuPrimer Pfx DNA polymerase (interpreted as comprising double-indexed libraries; and an error rate of no more than 2.25 x 10-10, claim 21) (pg. 5, col 1; last full paragraph, lines 1-3), wherein it is known that double-indexing keeps the sequencing error rate low as evidenced by Kircher (pg. 1, col 2; first full paragraph, lines 12-15).
Gansauge does not specifically exemplify a barcoded single-stranded adapter (claim 12, in part).
Regarding claim 12 (in part), Head teaches that to facilitate multiplexing, different barcoded adapters can be used with each sample, alternatively, barcodes can be intro-duced at the PCR amplification step by using different barcoded PCR primers to amplify different samples, wherein high quality reagents with barcoded adapters and PCR primers are readily available in kits from many vendors (interpreted as ligating a barcoded adapter, claim 1) (pg. 63, col 1, first full paragraph). Head teaches that the PCR reaction also adds index (barcode) sequences, where the preparation procedure improves on traditional protocols by combining DNA fragmentation, end-repair, and adaptor-ligation into a single step (pg. 63, col 1, second full paragraph).
It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of library construction for next generation sequencing as exemplified by Head, it would have been prima facie obvious before the effective filing date of the claimed invention to modify the method of preparing DNA sequencing libraries from a biological sample as disclosed by Gansauge to include the barcoded adaptors and/or indexed primers as taught by Head with a reasonable expectation of success in providing DNA libraries for high-throughput sequencing, for facilitating multiplexing, reducing errors, and/or in improving upon traditional protocols by combining DNA fragmentation, end-repair, and barcoded adaptor-ligation into a single step including for samples comprising low input of DNA.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly rejected under 35 USC §103 as obvious over the art.
Conclusion
Claims 1-4, 12-14 and 21 are rejected.
ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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