DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .2. This action is in response to the amendment filed on 07 November 2025. Applicant's arguments and amendments to the claims have been fully considered but do not place the application in condition for allowance. All rejections not reiterated herein are hereby withdrawn.
Claim Status
3. Claims 46, 48-50, and 53-65 are pending.
Claim 65 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 46, 48-50 and 53-64 read on the elected invention and have been examined herein.
Claim Interpretation
4. Claim 46 recites “each recognition site comprising a sequence of a minimum of about 10 nucleotides up to about 80 nucleotides.” Claim 59 recites “each recognition site comprising a sequence of a minimum of about 17 nucleotides up to about 80 nucleotides.”
In the reply of 07 November 2025, Applicant states:
“the claim phrase "a minimum of about 10 nucleotides up to about 80 nucleotides," when read in light of the specification defining the term "about," as acknowledged by the Office Action, as "containing the stated number of bases or base-pairs with a variation of 0-10% around the value (X 10%)" informs those skilled in the art with reasonable certainty about the length of the claimed recognition sites-a minimum of 9 and up to 88 nucleotides (where "about 10" contains 9-11 nucleotides and "about 80" contains 72-88 nucleotides, respectively). Therefore, one skilled in the art would readily recognize that "a minimum of about 10 nucleotides up to about 80 nucleotides" does not encompass 7 or 8 nucleotides as alleged in the Office Action. Similarly, a skilled artisan would recognize that recognition sites of claims 59-64 reciting "a minimum of about 17 nucleotides up to about 80 nucleotides" contains between 16 and 88 nucleotides (where "about 17" contains 15.3-18.7 nucleotides and "about 80" contains 72-88 nucleotides, respectively).”
Consistent with Applicant’s arguments, the definition in the specification at para [0047] for “about” with respect to polynucleotides is considered to apply to the endonuclease recognition site. Thus, claims 46, 48 and 53-58 are considered to require that the recognition site for the endonuclease consists of a minimum of 9 nucleotides up to 88 nucleotides and claims 59-64 are considered to require that the recognition site for the endonuclease consists of a minimum of 16 nucleotides up to 88 nucleotides.
Modified Claim Rejections - 35 USC § 103
5. 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 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 46, 48-50, and 53-64 is/are rejected under 35 U.S.C. 103 as being unpatentable over Stemeers et al (U.S. 20130316917; cited in the IDS) in view of Wang et al (U.S. 20180002706) and Zeiner et al (U.S. 20140357523)
Stemeers teaches a method for capturing a target genomic region from genetic material, the method comprising: cleaving a target genomic region at a 5’ end and a 3’ end with one or more endonucleases, including endonucleases that have recognition sites of 7 base pairs and rare cutters; denaturing the cleaved genetic material to form single-stranded target DNA; and capturing the single-stranded target DNA using a selector probe having sequences at the 5’ and 3’ ends that hybridize to the 5’ and 3’ ends of the single-stranded target DNA (see, e.g., para [0095], [0111], [0117] and Figure 3).
“As a first step in the circularization reaction, the DNA sample is digested by restriction enzymes to generate target fragments with defined ends. The digested DNA sample can be then denaturated to allow the selector to hybridize to the restriction fragments and template ligation to the vector oligonucleotide, forming single-stranded circular DNA molecules. This step can be performed in a least two methods. In a first method the ends of a targeted restriction fragment hybridize to the appropriate selector probe, and the ends become juxtaposed to the vector oligonucleotide guided by the selector probe. Next, a ligase joins the restriction fragment to the vector oligonucleotide generating a circular DNA strand.”
As shown in Figure 3A, the 5’ and 3’ ends of the selector probe are target-specific and thereby hybridize to the 5’ and 3’ ends of the single-stranded target DNA (Figure 3B). Thus, the selector probe of Stemeers constitutes a “bridge oligo.”
Regarding the limitation that the bridge oligo is a single stranded oligonucleotide, Stemeers (para [0109]) teaches “selector probes comprise hybridization tags, e.g., target-complementary end-sequences, joined by a general linking sequence adapted to ligate templates and to direct circularization of target nucleic acids.” The selector probe (i.e., bridge oligo) having sequences at the 3' and 5' ends that hybridize to the sequences at the 3' and the 5' ends, respectively, of the single stranded target genomic region is thereby a single stranded oligonucleotide.
Stemeers does not teach methods that comprise cleaving the target genomic region with an endonuclease that has a recognition site that has a minimum length of at least 9 nucleotides (i.e., “about 10 nucleotides” - claims 46, 48-50 and 53-58) or a minimum length of 16 nucleotides (i.e., “about 17 nucleotides” - claims 59-64), particularly wherein the endonuclease is a programmable endonuclease, and specifically is a Cas9 endonuclease.
However, Wang teaches methods of cleaving target genomic DNA using Cas9 endonuclease (e.g., para [0004]). Wang teaches that “Commonly used restriction enzymes have six or eight bp recognition sequences, which have an occurrence frequency of one in every 4096 or 65536 bp, respectively, in a random sequence” (para [0065]). It is disclosed that this can be problematic when a restriction endonucleases are not suitable when a restriction site is not located in a target region or when multiple restriction sites are present in a target sequence due to the frequency at which the site may occur (para [0065]). It is disclosed that CRISPR-associated protein-9 (Cas9) cleavage can overcome these problems since this endonuclease is programmable and has a recognition site of about 20 bp (para [0071] and [0109]). For instance, Wang (para [0071]) states:
“The CRISPR-associated protein-9 (Cas9) is an endonuclease that cleaves a double-stranded DNA target site guided by a single guide RNA (sgRNA). A sgRNA is composed of a fusion of target-specific CRISPR-related sequence (crRNA) that is from the target sequence and a trans-activating CRISPR-related RNA (tracrRNA) sequence that is from the bacterial CRISPR system. A crRNA, also known as protospacer, is a sequence of usually 20-nucleotides.”
Wang concludes that Cas9 nuclease is highly efficient and can be used as a restriction enzyme to cleave DNA (para [0107]).
Further Zeiner teaches methods of fragmenting genomic DNA and detecting target polynucleotides comprising cleaving a target nucleic acid with CRISPR-associated Cas proteins and a plurality of, including at least two, Cas9-associated guide RNAs, wherein one guide RNA directs cleavage at the 5’ end of a target DNA and a second guide RNA directs cleavage at the 3’ end of the target DNA (e.g., para [0003], [0033], [0049], [0074] and [0088]). It is disclosed that the Cas9-associated guide RNAs may each be specific for a different, pre-defined, site in genomic DNA (para [0063]).
Zeiner (para [0049]) states:
“The guide RNAs used in the method may be designed so that they direct binding of the Cas9-gRNA complexes to pre-determined cleavage sites in a genome. In certain cases, the cleavage sites may be chosen so as to release a fragment that contains a region of unknown sequence, or a region containing a SNP, nucleotide insertion, nucleotide deletion, rearrangement, etc.”
Zeiner further teaches isolating, amplifying and/or sequencing the DNA fragments generated by the Cas9 cleavage (para [0054] and [0090]).
In view of the teachings of Wang and Zeiner, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Stemeers so as to have cleaved the target genomic DNA with a Cas9-gRNA endonuclease in place of a restriction endonuclease with a recognition site of 6- or 7-bp. One would have been motivated to have done so for the benefits disclosed by Wang and Zeiner of providing a method with high level of specificity of cleavage since the Cas9-gRNA endonuclease system recognizes target sequences of 20-bp (i.e., a minimum of about 10 or about 17 nucleotides) and thereby the recognition site occurs less frequently in the genome and the target recognition site can be easily customized for a target genomic region based on the selection of the single stranded guide RNA.
Regarding claims 48-50, modification of the method of Stemeers as set forth above would have resulted in a method in which the genomic DNA is cleaved with the programmable endonuclease of Cas9-gRNA endonuclease, wherein the Cas9-gRNA endonuclease comprises a first endonuclease that cuts DNA at a first recognition site based on a first RNA guide molecule having a sequence complementary to the first recognition site and a second programmable endonuclease that cuts DNA at a second recognition site based on a second RNA guide molecule having a sequence complementary to the second recognition site.
Regarding claim 53, in the embodiment disclosed by Stemeers at para [0109]), the region between the target-complementary end-sequences, including a “linking sequence” is considered to include a primer binding site sequence, since a primer can bind to this sequence.
Regarding claim 54, Stemeers teaches that the probe is attached to a biotin moiety (i.e., “biotinylated”; e.g., para [0080]).
Regarding claims 55-58, as discussed above, Stemeers teaches the embodiment in which a capture probe comprises a selector probe (i.e., bridge oligo) and a vector probe (para [0110] and Figure 3). As shown in Figure 3B I, the ends of the digested genomic DNA are indirectly ligated via ligation with the vector probe portion that is hybridized to the selector probe (bridge oligo). This results in a structure comprising a circularized single-stranded target DNA hybridized to the selector probe (bridge oligo). Thus, Stemeers teaches “ligating the free ends of the single stranded target genomic region hybridized to the bridge oligo to produce a single stranded circular target genomic region that is hybridized to the bridge oligo.” Note that steps e) and f) of claim 55 are optional and thereby need not occur. Further, step g) of analyzing the amplified target genomic region is also considered to be optional because the only amplification step recited in the claim occurs in optional step f). Alternatively, Stemeers teaches “In some embodiments, after the circularization reaction, linear sample DNA is degraded by exonucleolysis” (para [0111]. Thereby, Stemeers teaches degrading non-circularized genetic material. Stemeers teaches amplifying the circularized target DNA (e.g., para [0111] and [0114]) and also teaches sequencing the target genomic DNA (e.g., para [0006], [0016], [0060] and [0109]).
Regarding claim 56, Stemeers teaches that the circularized target DNA can be amplified by rolling circle replication, which is considered to be rolling circle amplification (e.g., para [0114] “Circular nucleic acid molecules can be amplified by a variety of methods, for example, rolling circle replication”).
Regarding claim 57, as discussed above, the analyzing step is considered to be optional since it depends on the optional amplifying step. Further, Stemeers teaches sequencing the Cas9-cleaved target genomic DNA (e.g., para [0006], [0016], [0060] and [0109]).
Regarding claim 58, Stemeers does not teach that sequencing the Cas9-cleaved target genomic DNA comprises nanopore sequencing.
However, as discussed above, Zeiner teaches sequencing the fragments generated by the Cas9 cleavage (para [0054] and [0090]), particularly using nanopore sequencing (para [0055]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Stemeers so as to have sequenced the Cas9-cleaved target genomic DNA region since Zeiner teaches that this is an effective means for sequencing DNA, and particularly DNA generated by Cas9-gRNA cleavage.
Regarding claims 59-64, as discussed above, modification of the method of Stemeers so as to have cleaved the target genomic DNA using the Cas9-gRNA endonuclease system would have resulted in a method in which the recognition site for the endonuclease is about 20 nucleotides in length, which is within the claimed minimum range of about 17 nucleotides up to about 80 nucleotides in length. Stemeers teaches that the method may be a multiplex method (e.g., para [0053], [0109] and [0113]). Stemeers also teaches the embodiment in which a capture probe (i.e., bridge oligo) comprises a selector probe and a vector probe (para [0110]). The capture probe itself also constitutes a bridge oligo. As shown in Figure 3, the vector probe portion of the capture probe (bridge oligo) is ligated to the 5’ and 3’ ends of the cleaved, single-stranded target DNA and this complex is subsequently amplified. Thus, this embodiment of Stemeers also includes a step of “capturing the plurality of target genomic regions in the single stranded form by hybridizing the plurality of target genomic region to a plurality of bridge oligos, wherein each bridge oligo comprises sequences at the 3' and 5' ends that hybridize to the 3' and 5' ends, respectively, of a target genomic region from the plurality of target genomic regions in single stranded form.”
Regarding claim 61, Stemeers teaches that the capture probe (i.e., bridge oligo) can be attached to an affinity tag, such as a biotin moiety so as to facilitate the separation of the affinity tagged nucleic acids from untagged molecules (para [0116-0117]). For instance, Stemeers states “The selector probe is ligated to the ssDNA. Biotin affinity moieties of the capture probes bind to streptavidin bound to beads. The beads are washed and unassociated nucleic acids are stringently washed from the beads. The washed nucleic acids are eluted from the beads.” (para [0117]). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have performed the method of Stemeers using a biotin-tagged selector probe / bridge oligo that was subsequently immobilized onto a solid support / streptavidin coated bead in order to have facilitated the separation of the cleaved target genomic DNA region bound to the capture probe / bridge oligo from unbound DNA and other reagents, thereby permitting the isolation of the cleaved target genomic DNA region bound to the capture probe / bridge oligo, as disclosed by Stemeers.
Regarding claims 62-64, Stemeers teaches amplifying the cleaved target genomic regions by PCR to form multiple copies of the target genomic region (e.g., para [0070], [0109], [0111] and [0113]).
Further, regarding claim 64, Stemeers teaches performing multiplex PCR using primers that are a universal primer pair and which bind to the “general primer-pair motif” present in the selector probe (e.g., Figure 3 and [0110-0111]).
Response to Remarks:
In the reply of 07 November 2025, Applicant traversed the previous rejection of the claims and stated that “Stemeers does not teach or suggest a bridge oligo that is a single-stranded oligonucleotide as required by claim 46.”
Applicant’s arguments have been fully considered but are not persuasive. In particular, Applicant states “Figure 3A shows a ‘selector probe comprising two oligonucleotides, one with two end sequences complementary to the target sequence separated by a general primer-pair motif and a vector oligo complementary to the general primer-pair motif. (Stemeers, page 11, paragraph 110 and Figure 3A).”
However, in Figure 3A, the single stranded oligonucleotide having “two end sequences complementary to the target sequence separated by a general primer-pair motif” and labeled therein as a selector probe is considered to be a bridge oligonucleotide. It itself is a single-stranded oligonucleotide that is then hybridized to the vector oligonucleotide. Para [0110] of Stemeers states:
“a selector probe comprises two oligonucleotides: one selector probe with two end sequences complementary to the target sequence to be selected for amplification, separated by a general primer-pair motif, and a vector oligonucleotide complementary to a general primer-pair motif (FIG. 3).”
The “one selector probe with two end sequences complementary to the target sequence to be selected for amplification, separated by a general primer-pair motif is a single-stranded oligonucleotide” is a single-stranded oligonucleotide. See Figure 3A in which the “Sector probe” is the bottom single-stranded DNA molecule with the “Target specific ends” as shown below:
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Regarding Applicant’s argument that para [0109] of Stemeers “does neither expressly nor inherently teach single-stranded oligonucleotides that are ligated to a template,” the teachings at para [0109] together with Figure 3 do establish that the oligonucleotide bridging/joining the cleaved genomic nucleic acid is single stranded. At para [0109], Stemeers states:
“In some embodiments, selector probes comprise hybridization tags, e.g., target-complementary end-sequences, joined by a general linking sequence adapted to ligate templates and to direct circularization of target nucleic acids.”
These teachings of Stemeers clearly establish that the method therein requires a single-stranded oligonucleotide with a 5’ and 3’ end that is complementary to a target nucleic (separated by a general primer sequence) wherein the single-stranded oligonucleotides “bridges” / joins / circularizes the target nucleic acid.
Note also that claim 46 requires only the steps of cleaving the target genomic region, denaturing the target genomic region into single-stranded form and capturing the target genomic region by hybridizing the target genomic region to a bridge oligo. The claim does not exclude a step in which the bridge oligo (“selector oligo” in this embodiment of Stemmers) becomes hybridized to a vector oligonucleotide.
Further, Applicant’s arguments are not persuasive as they pertain to claims 59-64 because these claims do not require that the bridge oligo is a single-stranded oligonucleotide. While claim 46 was amended to require that the bridge oligo is a single-stranded oligonucleotide, claims 59-64 were not amended to require this limitation. Claims 59-64 as broadly recited encompass methods in which the bridge oligo is a double-stranded oligonucleotide. As discussed in the rejection as it pertains to claims 59-64, the double-stranded capture oligonucleotide of Stemeers comprising the single-stranded selector probe and the vector oligonucleotide (Figure 3A) can also be viewed as a “bridge oligo.” As shown in Figure 3, the capture oligonucleotide binds to and captures a “plurality of target genomic regions in the single stranded form by hybridizing the plurality of target genomic region to a plurality of bridge oligos, wherein each bridge oligo comprises sequences at the 3' and 5' ends that hybridize to the 3' and 5' ends, respectively, of a target genomic region from the plurality of target genomic regions in single stranded form.”
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS 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 CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682