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 .
Application Status
This action is written in response to applicant’s correspondence received 16 March 2026. Claims 1-2, 4, 6, 8-9, 13, 16, 18, 20, 31-32, 36-37, and 41-46 are currently pending.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 102
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 (i.e., changing from AIA to pre-AIA ) 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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Crawford (WO2018/035062, published February 22, 2018, filed 14 August 2017) as evidenced by Sternberg (Biophysical Journal 106.2 (2014): 695a). This rejection is maintained.
Regarding claim 1, Crawford is drawn towards an invention concerned with a method of sample analysis (Abstract). Crawford teaches that the method comprises (a) digesting an end-blocked, phosphatase-treated, mixed nucleic acid sample (i.e., a protected polynucleotide sample that has its the 5’ ends dephosphorylated such that they’re unligatable (pg. 17)) with (b) a plurality of reprogrammed nucleic acid directed endonucleases (i.e., polynucleotide-guided effector proteins that utilize a guide RNA to target a nucleic acid of interest (pg. 11)) that target sequences of interest to produce a digested sample (pg. 1-2). Crawford teaches that two or more Cas9 cut sites may be present within the polynucleotide flanking a fragment comprising a region of interest that is to be amplified or sequenced (i.e., the Cas9 molecules may create opposing cuts on two or more sites determined by the sequences to which the two or more guide polynucleotides bind) (pg. 15; see FIG. 1). Crawford teaches that (c) the cleaved region of interest can be enriched and have adaptors attached to both opposing ends of the polynucleotide and not the protected ends of the protected polynucleotide (pg. 15; see Fig. 1). Crawford teaches that the region of interest may be longer than 1 kb in length (i.e., at least about 1000 nucleotides in length) (pg. 14-15). Crawford further teaches that “the guide RNAs/DNAs may be may be chosen to release fragments that are of a size that are suitable for size selection” (pg. 14).
Sternberg is drawn towards a study concerned with DNA interrogation by CRISPR-Cas9 endonucleases (Abstract). Sternberg teaches that Cas9 proteins can bind to a dsDNA molecule (i.e., a dsDNA molecule comprising a region of interest and a region of non-interest) and, following the addition of urea, release a downstream end of the dsDNA containing a PAM sequence (i.e., a region of interest) while the Cas9 remains bound to an upstream region of the dsDNA not comprising the PAM (i.e., a region of dsDNA not comprising a region of interest) (pg. 3; see Figure 2).
Therefore, in order for the fragment comprising a region of interest of Crawford to be released, the fragment must inherently comprise a PAM and the Cas9 must inherently remain attached to the region of dsDNA that does not comprise the PAM or region of interest, as evidenced by Sternberg. This is because, as taught by Sternberg, following dsDNA cleavage Cas9 inherently releases the region of dsDNA that comprises a PAM sequence and inherently remains attached to the remaining region of dsDNA that does not comprise the PAM sequence. Thus, the “released fragment” of Crawford must inherently comprise a Cas9 PAM sequence that is released from the dsDNA break while the Cas9 remains attached to the region not comprising the PAM and the region of interest.
Regarding claim 2, Crawford teaches that the polynucleotides were contacted with calf intestinal phosphatase (i.e., a dephosphorylase) for 30 minutes at 37 °C in order to dephosphorylate all 5’ DNA ends present (pg. 30).
Regarding claim 6, Crawford teaches the use of a Cas9 RNA-guided effector protein (pg. 11).
Regarding claim 8, Crawford teaches that the target polynucleotide may be double stranded cDNA (see FIG. 1).
Regarding claim 9, Crawford teaches that the Cas9 can cut both strands of a double stranded polynucleotide (see FIG. 1) to produce a blunt end or other endonucleases can be utilized to produce an overhang (pg. 12-13).
Regarding claims 16 and 41, Crawford teaches that the adapters can be ligated (i.e., covalently attached to) to the region of interest (pg. 1).
Regarding claim 18, Crawford teaches that the cleaved region of interest can be enriched and have adaptors attached to both opposing ends of the polynucleotide and not the protected ends of the protected polynucleotide (pg. 15; see Fig. 1).
Regarding claims 31-32, Crawford teaches that the two or more guide polynucleotides may be utilized to cleave multiple regions of interest from a dsDNA molecule such that the regions of interest becomes flanked by adapters (i.e., two or more guide polynucleotides are used in the method in order to attach adapters flanking two or more regions of interest) (see FIG. 1).
Regarding claim 36, Crawford teaches that the enriched fragments may be analyzed (i.e., characterized) (pg. 1-2).
Regarding claim 43, Crawford teaches that the guide sequences flanking the region of interest and are not located within the region of interest (see FIG. 1).
Regarding claims 45-46, Crawford teaches that the adapters are a pair of adapters that can be enriched by PCR and utilized to amplify the target region of interest (pg. 13, 15 )
Claim Rejections - 35 USC § 103
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.
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.
Claim(s) 4, 13, and 44 is/are rejected under 35 U.S.C. 103 as being unpatentable over Crawford (WO2018/035062, published February 22, 2018, filed 14 August 2017) as evidenced by Sternberg (Biophysical Journal 106.2 (2014): 695a) as applied to claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 above, and further in view of Callewaert (WO2017/162754 A1, published September 28, 2017). This rejection is maintained.
Regarding claims 4, 13, and 44, Crawford as evidenced by Sternberg anticipates claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 as described above.
Crawford as evidenced by Sternberg does not teach or suggest that the 3’ end of the polynucleotides are extended by adding a terminal transferase an a dNTP to the sample of polynucleotides (Claim 4). Crawford as evidenced by Sternberg does not teach or suggest that the adapter comprises a single T and the method further comprises contacting the sample prior to step c with a polymerase and dATP to add a single A tail to at least one of the cut ends in the target polynucleotide (Claim 13). Crawford as evidenced by Sternberg does not teach or suggest that the method comprises contacting the sample with a polymerase and dNTPs to fill in the overhang to produce a blunt end (Claim 44).
However, one of ordinary skill in the art would have considered the teachings of Callewaert as both references are common fields of endeavor pertaining to the amplification of nucleotide sequences of interest.
Callewaert is drawn towards an invention concerned with preparing a collection of degraded DNA fragments isolated from a biofluid sample of an individual (Abstract). Callewaert teaches that Cas9 can be utilized to fragment dephosphorylated DNA into fragments that can be subsequently sequenced (pg. 18). Callewaert teaches that all possible overhangs (i.e., including a T overhang) present in the fragment can be filled in via the use of a DNA polymerase in combination with all dNTPs in order to generate a blunt end (pg. 18-19). Callewaert teaches that filling in the overhangs present in the fragmented DNA allows for the compatibility of the fragment with commercially used Illumina-kind adapters (pg. 21).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Crawford as evidenced by Sternberg such that the target region comprising an overhang was contacted with a dATP and a polymerase in order to generate a blunt end, as described by Callewaert. A person of ordinary skill in the art would have been motivated to do so in order to allow for the target region to become compatible with commercially used Illumina-kind adapters. A person of ordinary skill in the art would have had a reasonable expectation of success because both Crawford as evidenced by Sternberg and Callewaert are drawn towards methods that amplify fragmented DNA via the use of adapters.
Claim(s) 20 and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Crawford (WO2018/035062, published February 22, 2018, filed 14 August 2017) as evidenced by Sternberg (Biophysical Journal 106.2 (2014): 695a) as applied to claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 above, and further in view of Karamitros (Nucleic acids research 43.22 (2015): e152-e152). This rejection is maintained.
Regarding claims 20 and 42, Crawford as evidenced by Sternberg anticipates claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 as described above. Crawford further teaches that the adapters are a pair of adapters that can be enriched by PCR and utilized to amplify the target region of interest (i.e., the adapters are sequencing adapters) (pg. 13, 15 )
Crawford as evidenced by Sternberg does not teach or suggest that the adapter is an intermediate adapter and the method comprises attaching a further adapter to the intermediate adapter (Claim 20).
However, one of ordinary skill in the art would have considered the teachings of Karamitros as both references are common fields of endeavor pertaining to the use of adapters in PCR sequencing methods.
Karamitros is drawn towards a study concerned with the use of biotinylated PCR-generated baits in adapters (Abstract). Karamitros teaches that PCR baits are single stranded nucleic acids that can initiate PCR at a target nucleic acid sequence of interest that comprises a complementary sequence to the PCR bait (pg. 5; see Figure 1). Karamitros teaches the use of an intermediate adaptor that is attached to a biotin adapter that flank the PCR baits so that they can be attached to streptavidin beads (pg. 5; see Figure 1). Karamitros teaches that the use of the intermediate adapters allows for multiplexed nanopore sequencing of the target nucleic acid sequence (pg. 4).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the adapters of Crawford as evidenced by Sternberg for an adapter that comprises an intermediate adapter that is further attached to another adapter, as described by Karamitros. A person of ordinary skill in the art would have been motivated to do so in order to sequence the target nucleic acid via multiplexed nanopore sequencing. A person of ordinary skill in the art would have had a reasonable expectation of success because both Crawford as evidenced by Sternberg and Karamitros teach the attachment of adapters to a fragmented nucleic acid of interest in order to generate sequencing reads of the nucleic acid.
Claim(s) 37 is/are rejected under 35 U.S.C. 103 as being unpatentable over Crawford (WO2018/035062, published February 22, 2018, filed 14 August 2017) as evidenced by Sternberg (Biophysical Journal 106.2 (2014): 695a) as applied to claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 above, and further in view of Lu (Genomics, proteomics & bioinformatics 14.5 (2016): 265-279). This rejection is maintained.
Regarding claim 37, Crawford as evidenced by Sternberg anticipates claims 1-2, 6, 8-9, 16, 18, 31-32, 36, 41, 43, and 45-46 as described above. Crawford further teaches that nanopore sequencing methods may be utilized in the method to sequence the nucleic acid of interest (pg. 9, 16).
Crawford as evidenced by Sternberg does not teach or suggest that the sample obtained by claim 1 is contacted with a nanopore, a potential difference applied across the nanopore, and the presence or absence of an effect resulting from the interaction of the target polynucleotide with the nanopore is monitored in order to determine the presence or absence of the target polynucleotide (Claim 37).
However, one of ordinary skill in the art would have considered the teachings of Lu as both references are common fields of endeavor pertaining to the use of nanopore sequencing methods.
Lu is drawn towards a study concerned with a method of nanopore sequencing (Abstract). Lu teaches a method wherein the membrane of a nanopore can have a voltage applied to drive a DNA molecule through the pore, wherein an ion current flow can be measured (pg. 267, col 1, para 3). Lu teaches that when a DNA molecule passes through the nanopore, a change of the current in pattern or magnitude can be observed and characterized in order to determine if the DNA molecule is present (pg. 267, col 1, para 3). Lu teaches that the method can be utilized to equate raw current measurements with characteristics of the DNA sample (pg. 267).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the nanopore sequencing method of Crawford as evidenced by Sternberg for the nanopore sequencing method of Lu. A person of ordinary skill in the art would have been motivated to do so in order to equate current measurements with DNA sample characteristics. A person of ordinary skill in the art would have had a reasonable expectation of success because both Crawford as evidenced by Sternberg and Lu teach the use of nanopore sequencing methods that can be utilized to sequence target nucleic acids of interest.
Response to Arguments
Applicant's arguments filed 16 March 2026 have been fully considered but they are not persuasive.
Applicant alleges that the examiner has not provided evidence demonstrating that the Cas9 of Crawford’s disclosed method, during normal and usual operation, must inherently remain attached to a region of dsDNA that does not contain a region of interest during aptamer attachment, as required by instant claim 1 (Remarks; pg. 9). Applicant alleges that the method of Crawford does not anticipate the instantly pending method because prior to adapter ligation, Crawford treats the samples with proteinase K, converted into sequencing libraries according to manufacturer’s instructions; therefore, the method of Crawford cannot teach a method wherein the claimed adapters are attached to the region of interest while the Cas9 molecules remained attached to the region of dsDNA that does not contain the region of interest (Remarks; pg. 9-10).
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., the requirement that the Cas9 remains attached to the region of dsDNA that does not contain the region of interest during the adapter attachment) are not recited in the rejected claim(s). 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, 26 USPQ2d 1057 (Fed. Cir. 1993).
It is noted that the claimed method present in instant claim 1 recites method steps (a)-(c) that are performed in sequential order. The method does not claim that step (c) (i.e., the attachment of an adapter to one or both of the two opposing cut ends in the target polynucleotide) happens simultaneously with step (b). Rather, given the claim’s broadest reasonable interpretation, the three individual method steps are performed in the disclosure of Crawford because, as described above, Crawford teaches that (a) the ends of the polynucleotides are protected as claimed; (b) two Cas9 molecules target and cleave nucleic acid sequences that flank a region of interest, wherein, as evidenced by Sternberg, the two Cas9 molecules remain attached to the regions of the polynucleotide that do not contain the region of interest as claimed; and (c) adapters are attached to both sides of the region of interest as claimed. The remaining claim limitations present in the claim (e.g., see lines 22-26 of claim 1), do not specify that the polynucleotide-guided effector proteins must remain attached during method step (c). Rather, the remaining claim limitations recite that the adapter does not attach to the protected ends of the polynucleotide and that the target polynucleotide is at least 1000 nucleotides in length. Accordingly, Applicant’s arguments with respect to the claimed method steps (b) and (c) happening simultaneously are not found persuasive.
Applicant alleges that Sternberg does not cure the deficiency of Crawford and that the attachment taught in Sternberg is irrelevant in light of Crawford’s method, which removes Cas9 before adapter ligation (Remarks; pg. 10).
This argument is not found persuasive because, as discussed above, the claimed method steps (b) and (c) do not happen simultaneously. Thus, the attachment taught in Sternberg is relevant evidence pointing to the fact that the Cas9s of Crawford would remain attached to the region of dsDNA that does not contain the region of interest prior to the adapter ligation, as currently claimed.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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