DETAILED ACTION
Status of the Claims
Claims 1-18, 24, and 26 are currently pending and are the subject of this Office Action.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/22/2022 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Rejections – 35 U.S.C. 103(a)
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.
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.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Franchini et al., Currin et al., Lebrigand et al., Vermaas et al., and NEB
Claims 1-9, 13, 16, 18, 24, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Franchini et al. (Molecular Ecology, 2017, 26(10): 2783-2795, and supplementary material, each cited in IDS of 11/22/2022) in view of Currin et al. (Synthetic Biology, 2019 4(1):ysz025) and Lebrigand et al. (bioRxiv preprint doi.org/10.110/831495), further in view of Vermaas et al. (U.S. PGPub 2018/0305751 A1, cited in IDS of 11/22/2022), and further in view of NEB (NEB Featured Article, 2019).
Regarding claim 1, Franchini discloses a pair of oligonucleotide adapters, wherein said pair comprises a first oligonucleotide adapter comprising
a top strand comprising 5' – N6 barcode sequence (e.g., in red as per Supplementary Table 3) – N4 sequence (e.g., TGCA to match the restriction enzyme PstI as per Supplementary Table 5) corresponding to a sticky end left by digestion by a first restriction enzyme - phosphate - 3' wherein at least one of the nucleotide(s) of the N6 barcode sequence immediately adjacent to the N4 sequence corresponding to the sticky end left by digestion by said first restriction enzyme is different to the corresponding nucleotide(s) of the recognition sequence of said first restriction enzyme (e.g., a G rather than a C as per Supplementary Table 3); and a bottom strand comprising 5' - phosphate – N6 barcode sequence complementary to the N6 barcode sequence of the top strand (e.g., in red as per Supplementary Table 3) - N4 unique molecular identifier (UMI) sequence (e.g., shown as NNNN in blue of Supplementary Table 3) - binding site for at least one oligonucleotide primer - 3'; and
a second oligonucleotide adapter comprising
a top strand comprising 5'- N2 sequence corresponding to sticky end left by a second restriction enzyme (e.g., as per Supplementary Table 3) – N6 barcode sequence (e.g., in red as per Supplementary Table 3) - 3' wherein at least one of the nucleotide(s) of the N6 barcode sequence immediately adjacent to the N2 sequence corresponding to the sticky end left by said second restriction enzyme is different to the corresponding nucleotide(s) of the recognition sequence of said second restriction enzyme (e.g., a G rather than a C as per Supplementary Table 3); and a bottom strand comprising 5' - binding site for at least one oligonucleotide primer - N4 unique molecular identifier (UMI) sequence (e.g., shown as NNNN in blue of Supplementary Table 3) – N6 barcode sequence complementary to the N8-24 barcode sequence of the top strand (e.g., in red as per Supplementary Table 3) - 3'.
However, it is noted that the reference discloses i5 and i7 barcode sequences that are six oligonucleotides in length, rather than the N8-24 as set forth in claim 1.
Franchini discloses “incorporating a phosphothioate bond at [the] 3’ ends to prevent unspecific/proofreading nuclease degradation” (e.g., as per the Adapter and primer design for quaddRAD section on p. 2787) and that the primers are purified away using AmpureXP beads (e.g., as per the Library preparation section on pp. 2787-2788), however, the reference is silent regarding the limitation wherein at least the 6 bases at the 3' terminal end of the bottom strand are each phosphorothioated, as set forth in claim 1.
Vermaas discloses purification of adapter-ligated libraries using oligonucleotides with three phosphorothioate binds at the 3’ ends (e.g., as per [0073]-[0074]) and subject to library clean-up using exonuclease digestion with at least one dsDNA specific nuclease and at least one ssDNA specific nuclease (e.g., as per [0092]-[0100]).
NEB states that in such circumstances “it is typically recommended that 3–6 [phosphorothioate] bonds be used to block exonuclease digestion” (e.g., as per p. 2, middle column).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to incorporate phosphorothioated bonds into at least the 6 bases at the 3' terminal end of the bottom strands, as per Vermaas and NEB, in the adapters of Franchini. One of ordinary skill in the art would have been motivated to do so since this allows for a quick and efficient library cleanup as per Vermaas at [0092]-[0100]. Further, as per MPEP 2143(I)(A), the rationale to support a conclusion that the 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 yielded nothing more than predictable results to one of ordinary skill in the art.
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since both NEB and Vermaas teach that phosophorothioate groups are common and can readily be incorporated during adapter synthesis from commercial sources.
Regarding claims 1, 3, and 7 and the requirements of N8-24 barcode and N4-24 UMI lengths, it is noted that the prior art discloses several possible lengths and that in accordance with MPEP 2144.05(I), in the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists. For example, Currin discloses using indexing barcodes that are 24 nucleotides in length (e.g., as per the Design of multiplexing primers section on p. 2) and Lebrigand discloses the use of UMIs that are longer than 10 nucleotides (e.g., as per Supplementary Notes on p. 8).
Regarding claim 2, Franchini discloses the above, wherein said oligonucleotide top strand of (a) and/or (c) further comprises a phosphate group at its 5' terminal end (e.g., as per Supplementary Table 3).
Regarding claim 4, Franchini discloses the above, wherein said N4-24 unique molecular identifier (UMI) sequence is a N4 unique molecular identifier (UMI) sequence (e.g., as per Supplementary Table 3).
Regarding claim 5, Franchini discloses the above, wherein said N8-24 barcode sequence is a N8 barcode sequence, and wherein said N4-24 unique molecular identifier (UMI) sequence is a N4 unique molecular identifier (UMI) sequence (e.g., as per Supplementary Table 3).
Regarding claim 6, Franchini discloses the above, wherein the binding site for at least one oligonucleotide primer of the bottom strand of strand of (a) and/or (c) comprises, or consists of, SEQ ID NO: 7, and wherein the binding site for at least one oligonucleotide primer of the bottom strand of strand of (b) and/or (d) comprises, or consists of SEQ ID NO: 8 (e.g., as per Supplementary Table 3).
Regarding claim 8, Franchini discloses the above, wherein the binding site for at least one oligonucleotide primer of the bottom strand of strand of (a) and/or (c) comprises, or consists of, SEQ ID NO: 9, and wherein the binding site for at least one oligonucleotide primer of the bottom strand of strand of (b) and/or (d) comprises, or consists of, SEQ ID NO: 10 (e.g., as per Supplementary Table 3).
Regarding claim 9, Franchini discloses the above, wherein the top strand N8-24 barcode sequence and the bottom strand N8-24 barcode sequence complementary to the N8-24 barcode sequence of the top strand are present as double stranded nucleic acid within the adapter (e.g., as per Supplementary Table 3).
Regarding claim 13, Franchini discloses a method of preparing a nucleic acid library from a sample comprising high molecular weight DNA (HMW DNA) comprising the steps of contacting said DNA with a first restriction enzyme and a second restriction enzyme; contacting said DNA with a pair of oligonucleotide adapters according to claim 1; contacting said DNA with at least one DNA ligase; and (iv) incubating to allow digestion of the DNA by said first restriction enzyme and second restriction enzyme, annealing of said oligonucleotide adapters to the digested DNA, and ligation of the annealed oligonucleotide adapters to the digested DNA by said at least one DNA ligase (e.g., as detailed above and/or Fig. 1).
Regarding claim 16, Vermaas discloses the above further comprising (v) contacting said DNA with at least one dsDNA specific nuclease and at least one ssDNA specific nuclease and incubating to allow digestion (e.g., as per [0092]-[0100]).
Regarding claim 18, Franchini discloses the above, further comprising purification of nucleic acid (e.g., as per the Library Preparation section).
Regarding claim 24, Franchini discloses a kit comprising a pair of oligonucleotide adapters according to claim 1, a DNA ligase and at least two restriction enzymes, each restriction enzyme leaving a different sticky end upon nucleic acid cleavage, and optionally one or more of buffer, one or more FFPE repair enzyme(s), one or more exonucleases (e.g., as detailed above and/or Fig. 1).
Regarding claim 26, Franchini discloses a method for generation of a DNA library, comprising the step of ligation of one or more adapter(s) according to claim 1 to one or more double stranded DNA fragment(s) comprising a single stranded overhang at each end of said fragment(s) (e.g., as detailed above and/or Fig. 1).
Franchini et al., Currin et al., Lebrigand et al., Vermaas et al., NEB, and Polisson et al.
Claims 1-13, 16, 18, 24, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Franchini et al. (Molecular Ecology, 2017, 26(10): 2783-2795, and supplementary material, each cited in IDS of 11/22/2022) in view of Currin et al. (Synthetic Biology, 2019 4(1):ysz025) and Lebrigand et al. (bioRxiv preprint doi.org/10.110/831495), further in view of Vermaas et al. (U.S. PGPub 2018/0305751 A1, cited in IDS of 11/22/2022), and further in view of NEB (NEB Featured Article, 2019), further in view of Polisson et al. (Nucleic Acids Research, 1992, 20(11):2888).
Franchini in view of Currin, Lebrigand, Vermaas, and NEB is relied on as above.
While Franchini discloses the use of PstI restriction enzyme with a recognition site 5'CTGCA/G3' and a N1-5 sequence that comprises TGCA, the reference is silent on the use of ApoI restriction enzyme with a recognition site 5'R/AATTY3' and a N1-5 sequence that comprises AATT, as set forth in claims 10-12.
Polisson discloses the use of ApoI restriction enzyme with a recognition site 5'R/AATTY3' and a N1-5 sequence that comprises AATT (e.g., as per the 2nd paragraph).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to use ApoI as per Polisson instead of MspI as per Franchini. In accordance with MPEP 2141 citing KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385,1395 (2007), "[s]imple substitution of one known element for another to obtain predictable results”, and as per MPEP 2143(I)(B), the rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.
Given the teachings of the prior art and the level of the ordinary skilled artisan at the time of the application’s effective filing date, it must be considered, absent evidence to the contrary, that said skilled artisan would have had a reasonable expectation of success in practicing the claimed invention.
Franchini et al., Currin et al., Lebrigand et al., Vermaas et al., and NEB
Claims 1-9, 13, 16-18, 24, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Franchini et al. (Molecular Ecology, 2017, 26(10): 2783-2795, and supplementary material, each cited in IDS of 11/22/2022) in view of Currin et al. (Synthetic Biology, 2019 4(1):ysz025) and Lebrigand et al. (bioRxiv preprint doi.org/10.110/831495), further in view of Vermaas et al. (U.S. PGPub 2018/0305751 A1, cited in IDS of 11/22/2022), and further in view of NEB (NEB Featured Article, 2019).
Franchini in view of Currin, Lebrigand, Vermaas, and NEB is relied on as above. However, the references are silent on the limitation of wherein said ssDNA specific nuclease comprises ExoI, as set forth in claim 17.
Vermaas specifically discloses that “[a]ny suitable exonuclease may be used” (e.g., as per [0097]) for library cleanup and specifically recommends Lambda exonuclease (e.g., as per [0098]).
New England Biolabs discloses that Exonuclease I and Exonuclease T have near identical nuclease properties:
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It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to use ExoI as per NEB instead of Exonuclease T as per Vermaas. In accordance with MPEP 2141 citing KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385,1395 (2007), "[s]imple substitution of one known element for another to obtain predictable results”, and as per MPEP 2143(I)(B), the rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.
Given the teachings of the prior art and the level of the ordinary skilled artisan at the time of the application’s effective filing date, it must be considered, absent evidence to the contrary, that said skilled artisan would have had a reasonable expectation of success in practicing the claimed invention.
Franchini et al., Currin et al., Lebrigand et al., Vermaas et al., NEBNext FFPE Repair Mix protocol, and NEB
Claims 1-9, 13-16, 18, 24, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Franchini et al. (Molecular Ecology, 2017, 26(10): 2783-2795, and supplementary material, each cited in IDS of 11/22/2022) in view of Currin et al. (Synthetic Biology, 2019 4(1):ysz025) and Lebrigand et al. (bioRxiv preprint doi.org/10.110/831495), further in view of Vermaas et al. (U.S. PGPub 2018/0305751 A1, cited in IDS of 11/22/2022), further in view of NEBNext FFPE Repair Mix protocol (2018) and further in view of NEB (NEB Featured Article, 2019).
Franchini in view of Currin, Lebrigand, Vermaas, and NEB is relied on as above, however, the references are silent on the limitations of the sample comprises formalin fixed paraffin embedded (FFPE) tissue and the use of NEBNext FFPE Repair mix, as set forth in claims 14-15.
New England Biolabs provides a kit for the extraction and repair of DNA from FFPE samples for library construction (e.g., throughout the document).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to use FFPE DNA as per NEBNext FFPE DNA Repair Mix protocol instead of fresh DNA samples as per Franchini. In accordance with MPEP 2141 citing KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385,1395 (2007), "[s]imple substitution of one known element for another to obtain predictable results”, and as per MPEP 2143(I)(B), the rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.
Given the teachings of the prior art and the level of the ordinary skilled artisan at the time of the application’s effective filing date, it must be considered, absent evidence to the contrary, that said skilled artisan would have had a reasonable expectation of success in practicing the claimed invention.
Conclusion
No claims are allowed.
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/JEREMY C FLINDERS/
Primary Examiner, Art Unit 1684