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 .
Response to Amendment
The amendment filed 02/02/2026 is acknowledged. Regarding the Office action mailed 11/03/2025:
The objection to claim 20 for typographical error is withdrawn in view of the amendment.
The rejection of claims 1, 2, 4-13 and 16-19 and the rejection of claims 20-35 under 35 USC 112(b) is maintained, though modified, as a result of the amendment.
The rejection of claims 1, 2, 6-13, 18 and 19 under 35 USC 103 over Pursiheimo (US 2022/0298566) in view of Larman (US 2023/0039899) is maintained and reiterated below.
The rejection of claims 1, 2, 6-13, 18 and 19 under 35 USC 103 over Pursiheimo (US 2022/0298569) in view of Larman (US 2023/0039899) is maintained and reiterated below.
The rejection of claims 20-22 and 24-35 under 35 USC 103 over Pursiheimo (US 2022/0298569) in view of Drmanac (Science 327:78-81 (2010)) and Drmanac Supplementary Material is maintained and reiterated below.
The rejection of claims 1, 2, 5-13, 17-18 under 35 USC 103 over Pursiheimo (US 2022/0298569) in view of Larman (US 2023/0039899), Drmanac (Science 327:78-81 (2010)) and Drmanac Supplementary Material is maintained and reiterated below.
The rejection of claim 16 under 35 USC 103 over Pursiheimo (US 2022/0298569) in view of Larman (US 2023/0039899), Drmanac (Science 327:78-81 (2010)), Drmanac Supplementary Material and Weng (US 2023/0265486) is maintained and reiterated below.
Applicant’s remarks will be addressed following the rejections.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 4-13 and 16-19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites, at step v-b, “bringing the one or more single-stranded concatemeric sequence in contact with a solid support, alternatively comprising a second capture moiety and allowing the first capture moiety in the concatemeric sequence and the second capture moiety to interact such that the hybridization complexes become linked to the solid support and separating the solid-support-linked hybridization complexes”. However, the “first capture moiety” is recited earlier in the claim (last three lines of step i) to be comprised by “at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex”. In the course of the claimed method, the first and second probes become ligated to form a “ligated ligation complex”, which is amplified by rolling circle amplification to form the single-stranded concatemeric sequence. That is, the ligated ligation complex serves as a template for rolling circle amplification; it does not itself become “part of” the single-stranded concatemeric sequence”. Therefore, the concatemeric sequence does not comprise the first capture moiety. Rather, the “first capture moiety” in this context appears to be for the purpose of capturing “hybridization complexes” (not the “single-stranded concatemeric sequence”) so as to separate them from other sample components (specification filed 11/02/2023, page 6, line 27 through page 7, line 2). However, this is disclosed as occurring prior to ligating the probes of the hybridization complex to form the template for rolling circle amplification. Therefore, Applicant has also created, by this amendment, what appears to be new matter. In any event, the concatemeric sequence of claim 1 does not comprise the first capture moiety.
Claims 2, 4-13 and 16-19 depend directly or indirectly from claim 1 and are rejected for the same reasons.
It is noted, however, that the rolling circle amplification can be carried out “using modified nucleotides containing capture moieties” (specification filed 11/02/2023, sentence spanning pages 7-8), which would provide for a capturing moiety within the concatemeric sequence itself. However, if Applicant wants to incorporate this optional feature into the claim, the claim will have to provide the step of using modified nucleotides containing capture moieties, and distinguish those from the “first capture moiety” recited in the claim already.
Claims 20-35 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 20 recites, at step v, “bringing the one or more single-stranded concatemeric sequence in contact with a solid support, alternatively comprising a second capture moiety and allowing the first capture moiety in the concatemeric sequence and the second capture moiety to interact such that the hybridization complexes becomes [sic, “become”] linked to the solid support and separating the solid-support-linked hybridization complexes”. However, the “first capture moiety” is recited earlier in the claim (last three lines of step i) to be comprised by “at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex”. In the course of the claimed method, the first and second probes become ligated to form a “ligated ligation complex”, which is amplified by rolling circle amplification to form the single-stranded concatemeric sequence. That is, the ligated ligation complex serves as a template for rolling circle amplification; it does not itself become “part of” the single-stranded concatemeric sequence”. Therefore, it is unclear how the concatemeric sequence could comprise the first capture moiety. Rather, the “first capture moiety” in this context appears to be for the purpose of capturing “hybridization complexes” (not the “single-stranded concatemeric sequence”) so as to separate them from other sample components (specification filed 11/02/2023, page 6, line 27 through page 7, line 2). However, this is disclosed as occurring prior to ligating the probes of the hybridization complex to form the template for rolling circle amplification. Therefore, Applicant has also created, by this amendment, what appears to be new matter. In any event, the concatemeric sequence of claim 20 does not comprise the first capture moiety.
Claims 21-35 depend directly or indirectly from claim 20 and are rejected for the same reasons.
It is noted, however, that the rolling circle amplification can be carried out “using modified nucleotides containing capture moieties” (specification filed 11/02/2023, sentence spanning pages 7-8), which would provide for a capturing moiety within the concatemeric sequence itself. However, if Applicant wants to incorporate this optional feature into the claim, the claim will have to provide the step of using modified nucleotides containing capture moieties, and distinguish those from the “first capture moiety” recited in the claim already.
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.
The factual inquiries 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.
Claims 1-2, 6-13, 18 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Pursiheimo (US 2022/0298566, previously cited) in view of Larman (US 2023/0039899, previously cited).
Regarding claim 1, Pursiheimo disclosed:
A method for the high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:
Para [0013]: “…a method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, the method comprising the steps of…”.
(i) providing for each target nucleotide sequence in each of the samples: a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,
Para [0014]: “…(i) providing for each target nucleotide sequence in each of the samples: a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex…”.
wherein the first probe comprises, starting from the 5' end of the molecule, a first bridge oligo-specific sequence, first universal sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of first probe;
Para [0015]: “…wherein the first probe comprises, starting from the 5’ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3’ end of first probe…”.
Para [0086]: “…the first probe includes, starting from the 5’ end of the molecule, optionally a 5’ phosphate, a first bridge oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target specific portion at its 3’ end…”.
and wherein the second probe comprises, starting from the 5' end of the molecule, a second target specific portion, optionally a second sequence barcode, second universal sequence, and a second bridge oligo-specific sequence at the 3' end of second probe;
Para [0016]: “…and wherein the second probe comprises, starting from the 5’ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3’ end of second probe…”.
Para [0086]: “…the second probe includes, starting from 5’ end of the molecule, optionally a 5’ phosphate, a second target specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridge oligo-specific sequence at its 3’ end…”.
and wherein the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;
Para [0017]: “…and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode…”.
and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;
Para [0018]: “…and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively…”.
and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease;
Para [0101]: “…at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease…”.
and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex, comprises a first capture moiety,
Para [0018]: “…and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety…”.
(ii) forming hybridization complexes…
Para [0019]: “…contacting, for each of the one or more target nucleotide sequence, the first probe and the second probe with, preferably for each of the samples in a separate tube, the bridge oligo or plurality of oligonucleotides capable of forming a bridge oligo complex and allow self-annealing into a plurality of ligation complexes…”;
Para [0020]: “…contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes…”;
Para [0021]: “…allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex…”.
(iii) ligating the probes in the one or more hybridization complexes to provide one or more ligated ligation complexes, using a ligase enzyme or enzymes or a combination of a ligase and a DNA polymerase,
Para [0024]: “…ligating the probes in the hybridization complexes to provide ligated ligation complexes…”.
Para [0082]: “After hybridization, the left and right probe are ligated chemically or enzymatically by a DNA ligase to form ligated ligation complex.”
(iv) amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase to form an amplified one or more single-stranded concatemeric sequence,
Para [0026]: “…(ix) amplifying nucleic acids from the one or more ligated ligation complexes…”.
Para [0101]-[0102]: “…step (ix) is performed by…(a) amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase…”.
and performing one of: (v-a) optionally subjecting the amplified one or more single-stranded concatemeric sequence obtained in step (iv) to annealing with a specific oligonucleotide containing the recognition sequence for the endonuclease wherein the specific oligonucleotide anneals with the recognition sequence of the first probe or the second probe or the bridge oligo or bridge oligo complex to form annealed complexes containing the recognition site for the endonuclease;
Para [0103]: “…optionally, subjecting the amplified one or more single-stranded concatemeric sequence obtained to annealing with a specific oligonucleotide containing said recognition sequence wherein said specific oligonucleotide anneals with said recognition sequence such that a recognition site for the said endonuclease is obtained…”.
and…cleaving the annealed complexes with said endonuclease to form nucleic acid fragments;
Para [0104]: “…optionally, cleaving the…annealed complexes obtained with said endonuclease…”.
(vi) subjecting nucleic acid fragments obtained in step (v-a)…to high-throughput sequencing technology to determine the barcode sequence(s);
Para [0027]: “…(x) subjecting the nucleic acids obtained in step (ix) to high-throughput sequencing technology to determine the barcode sequence(s)…”.
and (vii) identifying the presence and/or number of the target nucleotide sequence in each of the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode.
Para [0028]: “…identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode…”.1
Regarding claim 2, Pursiheimo disclosed (para [0054]): “…the plurality of samples includes a blood sample, a saliva sample, a urine sample or a feces sample…”.
Regarding claim 6, Pursiheimo disclosed (para [0213]): “The 3′ end of the third bridge oligo optionally includes a phosphate (or other cleavable) moiety…”.
Regarding claim 7, Pursiheimo disclosed (para [0137]): “…the 3′ end of the first probes or the 5′ end of the second probes, or both, are modified to permit chemical ligation of the first probes to the second probes.”
Regarding claim 8, Pursiheimo disclosed (para [0141]): “…the bridging portion of the first probe or the second probe, or both, comprise(s) chemically modified bases to permit improved binding to the bridge oligo or bridge oligo complex.”
Regarding claim 9, Pursiheimo disclosed (para [0091]): “The first target specific portion, the second target specific portion, the first bridge oligo-specific sequences, and/or the second bridge oligo-specific sequences, preferably contain independently from one another at least one chemically modified nucleotide to increase probe binding.”
Regarding claim 10, Pursiheimo disclosed (para [0104]): “Suitable strand-displacing polymerases include phi29 polymerase or Bst polymerase.”
Regarding claim 11, Pursiheimo disclosed (para [0109]): “Regardless of the method of amplification in step (ix), in some embodiment, the nucleotide molecules (RNA molecules, DNA molecules or cDNA molecules) are (further) amplified with a first primer and a second primer to provide an amplification product. Preferably a universal first primer and a universal second primer are used, reverse complementary to a first or second universal sequence present in the ligated complexes.”
Regarding claim 12, Pursiheimo disclosed (para [0110]): “…the genetic target enumeration is permitted by counting the number of molecular barcodes per target and per sample.”
Regarding claim 13, Pursiheimo disclosed (para [0130]): “In a preferred embodiment, for two or more samples or for two or more locus/allele combinations, barcode sequences are used to genotype the samples for one or more sequences and/or polymorphisms, such as SNPs and/or indels.”
Regarding claim 18, Pursiheimo disclosed (para [0025]): “…pooling the ligated ligation complexes from the plurality of samples…”.
Regarding claim 19, Pursiheimo disclosed cleaving the annealed complexes (para [0104]).
The only reason Pursiheimo does not anticipate is:
In the specific embodiment wherein the rolling circle amplification product is annealed to oligos to create a recognition sequence for the endonuclease, and the annealed complexes are cleaved, thereby producing fragments of the rolling circle amplification product, Pursiheimo did not explicitly state that these fragments were subjected to sequencing. Rather, the discussion merely ended with the cleavage step.2
Pursiheimo did not disclose forming the hybridization complexes as recited in the claim, whereby the probes were contacted with the target sequence first, followed by contacting that complex with the bridge oligo. Rather, Pursiheimo disclosed contacting the probes with the bridge oligo first, followed by contacting that complex with the target sequence.
However, as regards difference (i), it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to sequence those fragments, because the whole point of Pursiheimo’s disclosure was for sequencing; see Abstract: “The present invention disclosure relates to a next generation DNA sequencing method and use for accurate and massively parallel quantification of one or more nucleic acid targets…”. See also para [0027]: “…(x) subjecting the nucleic acids obtained in step (ix) to high-throughput sequencing technology to determine the barcode sequence(s)…”. In the embodiment discussed at para [0101]-[0105], step (ix) is performed by rolling circle amplification (RCA). The concatemeric sequence obtained by RCA is optionally annealed to oligonucleotide containing the site for the endonuclease (forming a complete double-stranded endonuclease cleavage site) and cleaved. Para [0105] disclosed that, in some embodiments no cleavage is performed and the sequencing step (x) is performed on the concatemeric sequence itself. However, it would have been obvious that, in cases where the cleavage step was performed, sequencing would still be carried out, but instead of sequencing the concatemeric sequence, the cleavage product of the concatemeric sequence would be performed so as to fulfill step (x) of Pursiheimo’s method, which required high-throughput sequencing.
As regards difference (ii), Larman disclosed an alternative technique in which two probes were first annealed to a target sequence, followed by annealing of a bridge oligo to the probes; see Fig. 2 and para [0034]. While Larman went on to do something else with the resulting product of rolling circle amplification (i.e. annealing detection probes, rather than cleaving into fragments and sequencing), Larman showed that the probes could be annealed to the target first, followed by annealing of a bridge oligo to create the circular template to be used for rolling circle amplification.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Pursiheimo by contacting the probes to the target sequence first, followed by annealing the bridge oligo to the probes, since Larman showed this to be a workable alternative order of the steps. This difference amounts to combining prior art elements (i.e. particular steps) according to known techniques to yield predictable results.
Claim(s) 1, 2, 6-13, 18 and 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pursiheimo (US 2022/0298569, hereinafter “Pursiheimo2”) in view of Larman (US 2023/0039899, previously cited).3 This rejection addresses option v-a of claim 1.
Regarding claim 1, Pursiheimo2 disclosed:
A method for the high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:
Para 0014: “a method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, the method comprising the steps of:”
(i) providing for each target nucleotide sequence in each of the samples: a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,
Para 0015-0016: “(i) providing for each target nucleotide sequence in each of the samples…a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,”
wherein the first probe comprises, starting from the 5' end of the molecule, a first bridge oligo-specific sequence, first universal sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of first probe;
Para 0017: “wherein the first probe comprises, starting from the 5′ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3′ end of first probe;”
Para 0079: “In certain embodiments, the first probe includes, starting from the 5′ end of the molecule, optionally a 5′ phosphate, a first bridge oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target specific portion at its 3′ end.”
and wherein the second probe comprises, starting from the 5' end of the molecule, a second target specific portion, optionally a second sequence barcode, second universal sequence, and a second bridge oligo-specific sequence at the 3' end of second probe;
Para 0018: “and wherein the second probe comprises, starting from the 5′ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3′ end of second probe”
Para 0079: “In certain embodiments, the second probe includes, starting from 5′ end of the molecule, optionally a 5′ phosphate, a second target specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridge oligo-specific sequence at its 3′ end.”
and wherein the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;
Para 0019: “and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode”
and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;
Para 0020: “and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively”
and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease;
Para 0021: “and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease”
and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex, comprises a first capture moiety,
Para 0022: “and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety”
(ii) forming hybridization complexes
Para 0023-0025: “(ii) contacting, for each of the one or more target nucleotide sequence, the first probe and the second probe with, preferably for each of the samples in a separate tube, the bridge oligo or plurality of oligonucleotides capable of forming a bridge oligo complex and allow self-annealing into a plurality of ligation complexes…(iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes…(iv) allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex”
(iii) ligating the probes in the one or more hybridization complexes to provide one or more ligated ligation complexes, using a ligase enzyme or enzymes or a combination of a ligase and a DNA polymerase,
Para 0027: “ligating the probes in the hybridization complexes to provide ligated ligation complexes”
Para 0075: “After hybridization, the left and right probe are ligated chemically or enzymatically by a DNA ligase to form ligated ligation complex.”
Para 0179: “FIG. 2B illustrates gap filling between the first probe and the second probe according to an embodiment herein. Here, the bridge oligo contains Gap1 between the bridge sequence 1 (228), and bridge sequence 2 (224). Gap2 is formed between the target binding parts of probes 1 and 2 (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. For this process a mixture of Stoffel fragment, Taq polymerase or Phusion polymerase, and DNA ligase such as Ampligase can be used. The polymerase adds nucleotides (a) complimentary to the universal bridge oligo sequence and (b) complimentary to the target sequence and thereby fills in the two gaps i.e. gap 1 and gap 2 between the first probe and the second probe, and the subsequent action of the DNA ligase results in ligation of the left probe and the right probe complementary to the bridge oligo and the target sequence, into a circular complex.”
(iv) amplifying nucleic acids from the one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase to form an amplified one or more single-stranded concatemeric sequence
Para 0029: “amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase”
(v-a) optionally subjecting the amplified one or more single-stranded concatemeric sequence obtained in step (iv) to annealing with a specific oligonucleotide containing the recognition sequence for the endonuclease wherein the specific oligonucleotide anneals with the recognition sequence of the first probe or the second probe or the bridge oligo or bridge oligo complex to form annealed complexes containing the recognition site for the endonuclease;
Para 0030: “optionally subjecting the amplified one or more single-stranded concatemeric sequence obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease wherein the oligonucleotide anneals with the recognition sequence specified in step (i) such that a recognition site for the endonuclease is obtained”
and cleaving the amplified single-stranded concatemeric sequence obtained in step (iv) or cleaving the annealed complexes with said endonuclease to form nucleic acid fragments;
Para 0031: “optionally cleaving the single-stranded concatemeric sequence obtained in step (viii) or the annealed complexes obtained in step (ix) with said endonuclease”
(vi) subjecting nucleic acid fragments obtained in step (v-a) or the one or more amplified single-stranded concatemeric sequence obtained in step (v-b) to high-throughput sequencing technology to determine the barcode sequence(s);
Para 0032: “subjecting the nucleic acid fragments obtained in step (x) or the concatemeric sequence obtained in step (viii) to high-throughput sequencing technology to determine the barcode sequence(s)”
and (vii) identifying the presence and/or number of the target nucleotide sequence in each of the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode.
Para 0033: “identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode”
Regarding claim 2, Pursiheimo2 disclosed (para [0043]): “…the plurality of samples includes a blood sample, a saliva sample, a urine sample or a feces sample…”.
Regarding claim 6, Pursiheimo2 disclosed (para [0135]-[0138]): “the bridge oligo comprises…(i) one to five 3′ protruding bases, and/or…(ii) 3′ phosphate, and/or…(iii) one or more phosphorothioate modifications within three positions from the 3′ end.”
Regarding claim 7, Pursiheimo2 disclosed (para [0139]): “…wherein the 3′ end of the first probe or the 5′ end of the second probe, or both, are modified to permit chemical ligation of the first probe to the second probe.”
Regarding claim 8, Pursiheimo2 disclosed (para [0140]): “…wherein the bridging portion of the first probe or the second probe, or both, comprise(s) chemically modified bases to permit improved binding to the bridge oligo.”
Regarding claim 9, Pursiheimo2 disclosed (para [0141]): “wherein the first target specific portion, the second target specific portion, the first bridge oligo-specific sequences, and/or the second bridge oligo-specific sequences, contain independently from one another, one or more chemically modified nucleotide.”
Regarding claim 10, Pursiheimo2 disclosed (para [0142]): “…using a phi29 polymerase or a Bst polymerase.”
Regarding claim 11, Pursiheimo2 disclosed (para [0143]): “…wherein a PCR amplification is performed between step (x) and step (xi) using primers which bind to universal parts of the first and second probes, wherein said primers optionally include adapters for subsequent sequencing in step (xi).”
Regarding claim 12, Pursiheimo2 disclosed (para [0144]): “…wherein genetic target enumeration is permitted by counting the number of molecular barcodes per target and per sample.”
Regarding claim 13, Pursiheimo2 disclosed (para [0145]): “…wherein for two or more samples or for two or more locus/allele combinations, barcode sequences are used to genotype the sample(s) for one or more sequences and/or polymorphisms, such as SNPs and/or indels.”
Regarding claim 18, Pursiheimo2 disclosed (para [0028]): “…pooling the ligated ligation complexes from the plurality of samples…”.
Regarding claim 19, Pursiheimo2 disclosed cleaving the annealed complexes (para [0031]).
Pursiheimo2 did not disclose forming the hybridization complexes as recited in the claim, whereby the probes were contacted with the target sequence first, followed by contacting that complex with the bridge oligo. Rather, Pursiheimo2 disclosed contacting the probes with the bridge oligo first, followed by contacting that complex with the target sequence.
Larman disclosed an alternative technique in which two probes were first annealed to a target sequence, followed by annealing of a bridge oligo to the probes; see Fig. 2 and para [0034]. While Larman went on to do something else with the resulting product of rolling circle amplification (i.e. annealing detection probes, rather than cleaving into fragments and sequencing), Larman showed that the probes could be annealed to the target first, followed by annealing of a bridge oligo to create the circular template to be used for rolling circle amplification.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Pursiheimo2 by contacting the probes to the target sequence first, followed by annealing the bridge oligo to the probes, since Larman showed this to be a workable alternative order of the steps. This difference amounts to combining prior art elements (i.e. particular steps) according to known techniques to yield predictable results.
Claim(s) 20-22, 24-35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pursiheimo (US 2022/0298569, hereinafter “Pursiheimo2”) in view of Drmanac (Science 327:78-81 (2010)) and Drmanac Supplementary Material [online] [retrieved on 30 October 2025] retrieved from https://www.science.org/doi/full/10.1126/science.1181498 .4
Regarding claim 1, Pursiheimo2 disclosed:
A method for the high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:
Para 0014: “a method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, the method comprising the steps of:”
(i) providing for each target nucleotide sequence in each of the samples: a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,
Para 0015-0016: “(i) providing for each target nucleotide sequence in each of the samples…a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,”
wherein the first probe comprises, starting from the 5' end of the molecule, a first bridge oligo-specific sequence, first universal sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of first probe;
Para 0017: “wherein the first probe comprises, starting from the 5′ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3′ end of first probe;”
Para 0079: “In certain embodiments, the first probe includes, starting from the 5′ end of the molecule, optionally a 5′ phosphate, a first bridge oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target specific portion at its 3′ end.”
and wherein the second probe comprises, starting from the 5' end of the molecule, a second target specific portion, optionally a second sequence barcode, second universal sequence, and a second bridge oligo-specific sequence at the 3' end of second probe;
Para 0018: “and wherein the second probe comprises, starting from the 5′ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3′ end of second probe”
Para 0079: “In certain embodiments, the second probe includes, starting from 5′ end of the molecule, optionally a 5′ phosphate, a second target specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridge oligo-specific sequence at its 3′ end.”
and wherein the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;
Para 0019: “and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode”
and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;
Para 0020: “and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively”
and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex, comprises a first capture moiety,
Para 0022: “and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety”
(ii) forming hybridization complexes:
(ii-a) by contacting, for each of the one or more target nucleotide sequence, the first probe, the second probe, and the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex and allowing self-annealing into a plurality of ligation complexes;
Para 0023: “(ii) contacting, for each of the one or more target nucleotide sequence, the first probe and the second probe with, preferably for each of the samples in a separate tube, the bridge oligo or plurality of oligonucleotides capable of forming a bridge oligo complex and allow self-annealing into a plurality of ligation complexes
contacting nucleic acids present in each of the plurality of samples to be tested for the one or more target nucleotide sequences with the plurality of ligation complexes;
Para 0024: “contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes”
and allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe from the plurality of ligation complexes to hybridize to essentially adjacent sections on the one or more target nucleotide sequences of each of the plurality of samples, thereby forming one or more first hybridization complexes;
Para 0025: “allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex”
(iii) ligating the probes in the one or more hybridization complexes to provide one or more ligated ligation complexes, using a ligase enzyme or enzymes or a combination of a ligase and a DNA polymerase,
Para 0027: “ligating the probes in the hybridization complexes to provide ligated ligation complexes”
Para 0075: “After hybridization, the left and right probe are ligated chemically or enzymatically by a DNA ligase to form ligated ligation complex.”
Para 0179: “FIG. 2B illustrates gap filling between the first probe and the second probe according to an embodiment herein. Here, the bridge oligo contains Gap1 between the bridge sequence 1 (228), and bridge sequence 2 (224). Gap2 is formed between the target binding parts of probes 1 and 2 (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. For this process a mixture of Stoffel fragment, Taq polymerase or Phusion polymerase, and DNA ligase such as Ampligase can be used. The polymerase adds nucleotides (a) complimentary to the universal bridge oligo sequence and (b) complimentary to the target sequence and thereby fills in the two gaps i.e. gap 1 and gap 2 between the first probe and the second probe, and the subsequent action of the DNA ligase results in ligation of the left probe and the right probe complementary to the bridge oligo and the target sequence, into a circular complex.”
(iv) amplifying nucleic acids from the one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase to form an amplified one or more single-stranded concatemeric sequences
Para 0029: “amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase”
(vi) subjecting the…amplified one or more single-stranded concatemeric sequences to high-throughput sequencing technology to determine the barcode sequence(s);
Para 0032: “subjecting the…concatemeric sequence obtained in step (viii) to high-throughput sequencing technology to determine the barcode sequence(s)”
and (vii) identifying the presence and/or number of the target nucleotide sequence in each of the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode.
Para 0033: “identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode”
Regarding claim 21, Pursiheimo2 disclosed (para [0043]): “…the plurality of samples includes a blood sample, a saliva sample, a urine sample or a feces sample…”.
Regarding claim 22, Pursiheimo2 disclosed (para [0022]): “…optionally, at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety…”.
Regarding claim 24, Pursiheimo2 disclosed (para [0135]-[0138]): “the bridge oligo comprises…(i) one to five 3′ protruding bases, and/or…(ii) 3′ phosphate, and/or…(iii) one or more phosphorothioate modifications within three positions from the 3′ end.”
Regarding claim 25, Pursiheimo2 disclosed (para [0139]): “…wherein the 3′ end of the first probe or the 5′ end of the second probe, or both, are modified to permit chemical ligation of the first probe to the second probe.”
Regarding claim 26, Pursiheimo2 disclosed (para [0140]): “…wherein the bridging portion of the first probe or the second probe, or both, comprise(s) chemically modified bases to permit improved binding to the bridge oligo.”
Regarding claim 27, Pursiheimo2 disclosed (para [0141]): “wherein the first target specific portion, the second target specific portion, the first bridge oligo-specific sequences, and/or the second bridge oligo-specific sequences, contain independently from one another, one or more chemically modified nucleotide.”
Regarding claim 28, Pursiheimo2 disclosed (para [0142]): “…using a phi29 polymerase or a Bst polymerase.”
Regarding claim 29, Pursiheimo2 disclosed (para [0143]): “…wherein a PCR amplification is performed between step (x) and step (xi) using primers which bind to universal parts of the first and second probes, wherein said primers optionally include adapters for subsequent sequencing in step (xi).”
Regarding claim 30, Pursiheimo2 disclosed (para [0144]): “…wherein genetic target enumeration is permitted by counting the number of molecular barcodes per target and per sample.”
Regarding claim 31, Pursiheimo2 disclosed (para [0145]): “…wherein for two or more samples or for two or more locus/allele combinations, barcode sequences are used to genotype the sample(s) for one or more sequences and/or polymorphisms, such as SNPs and/or indels.”
Regarding claim 32, Pursiheimo2 disclosed (para [0083]): “A first capture moiety, when used herein, refers to a moiety, such as a chemical group, which allows the probe, ligation complex or hybridization complex to be captured by, i.e. bound to, a second capture moiety which is linked to a solid support.”
Regarding claim 33, Pursiheimo2 disclosed (para [0026]): “optionally, bringing the hybridization complex in contact with a solid support comprising a second capture moiety, allowing the first capture moiety and the second capture moiety to interact such that the hybridization complexes become linked to the solid support and separating the solid-support-linked hybridization complexes from components of the samples that are not linked to the solid-support”
Regarding claim 35, Pursiheimo2 disclosed (para [0028]): “…pooling the ligated ligation complexes from the plurality of samples…”.
While Pursiheimo2 disclosed subjecting the concatemeric sequence to sequencing as discussed above, Pursiheimo2 did not disclose
(v) bringing the amplified one or more single-stranded concatemeric sequence in contact with a solid support…such that the amplified one or more single-stranded concatemeric sequence become linked to the solid support and separating the solid-support-linked amplified one or more single-stranded concatemeric sequences from components of the samples that are not linked to the solid support…using a solid support capable of binding modified or non-modified DNA with a high affinity
as recited in claim 20.
Also, while Pursiheimo2 disclosed capture of either “ligation complexes” (complexes of the probes and bridging oligo/oligo complex) or “hybridization complexes” (complexes of the probes, the bridging oligo/oligo complex, and target) as discussed for claims 32 and 33, Pursiheimo2 only taught the “separating” aspect in the case of the “hybridization complexes”, not for the “ligation complexes” as recited in claim 32. Pursiheimo did not teach capturing and separating “ligated ligation complexes” as recited in claim 34.
Drmanac taught a method for high-throughput sequencing concatemeric products of rolling circle amplification, in which the RCA products (which Drmanac called DNBs—DNA nanoballs) were brought in contact with a solid support, such that the amplified one or more single-stranded concatemeric sequence become linked to the solid support; see Fig. 1, panel C. As discussed in the Drmanac Supplementary Material, the solid support comprised “aminosilane features patterned onto the substrate serve as binding sites for individual DNBs”, and after binding the substrate was “rinsed to neutralize pH and remove unbound DNBs” (pages 6-7, section entitled “DNB array manufacturing”). The term “high affinity” is not defined in the specification as requiring any particular binding strength. Drmanac’s bound DNBs were able to remain bound following rinsing that removed unbound DNBs, and this falls within the broadest reasonable interpretation of “high affinity”.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Pursiheimo2 by using Drmanac’s sequencing technique for RCA products, because Drmanac taught this sequencing method “achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs” and offered high accuracy, affordable cost and scalability (abstract). The rejection is not positing replacing Pursiheimo’s entire method with Drmanac’s, but rather only sequencing the RCA products produced by Pursiheimo’s method using the patterned nanoarray technique of Drmanac, which “increased DNA content per array and image information density relative to random genomic DNA arrays” (page 78, third column, lines 12-15).
In addition, as Pursiheimo2 taught capturing and separating “hybridization complexes”, and taught capturing “ligation complexes”, it would have been obvious to perform the separation in the context of “ligation complexes” as well as capturing and separating “ligated ligation complexes” as recited in claims 32 and 34, respectively, for the same purpose, i.e. to separate non-productive components of the reaction from productive components.
Claim(s) 1, 2, 5-13, 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pursiheimo (US 2022/0298569, hereinafter “Pursiheimo2”) in view of Larman (US 2023/0039899, previously cited), Drmanac (Science 327:78-81 (2010)) and Drmanac Supplementary Material [online] 2010 [retrieved on 30 October 2025] retrieved from https://www.science.org/doi/full/10.1126/science.1181498.5 This rejection addresses option v-b of claim 1.
Regarding claim 1, Pursiheimo2 disclosed:
A method for the high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:
Para 0014: “a method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, the method comprising the steps of:”
(i) providing for each target nucleotide sequence in each of the samples: a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,
Para 0015-0016: “(i) providing for each target nucleotide sequence in each of the samples…a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex,”
wherein the first probe comprises, starting from the 5' end of the molecule, a first bridge oligo-specific sequence, first universal sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of first probe;
Para 0017: “wherein the first probe comprises, starting from the 5′ end of the molecule, a first bridge oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3′ end of first probe;”
Para 0079: “In certain embodiments, the first probe includes, starting from the 5′ end of the molecule, optionally a 5′ phosphate, a first bridge oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target specific portion at its 3′ end.”
and wherein the second probe comprises, starting from the 5' end of the molecule, a second target specific portion, optionally a second sequence barcode, second universal sequence, and a second bridge oligo-specific sequence at the 3' end of second probe;
Para 0018: “and wherein the second probe comprises, starting from the 5′ end of the molecule, a second target specific portion, optionally a second sequence barcode, and a second bridge oligo-specific sequence at the 3′ end of second probe”
Para 0079: “In certain embodiments, the second probe includes, starting from 5′ end of the molecule, optionally a 5′ phosphate, a second target specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridge oligo-specific sequence at its 3′ end.”
and wherein the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode;
Para 0019: “and wherein the bridge oligo or bridge oligo complex contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and optionally a third barcode”
and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively;
Para 0020: “and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively”
and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease;
Para 0021: “and wherein at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a recognition sequence for an endonuclease”
and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex, comprises a first capture moiety,
Para 0022: “and wherein, optionally, at least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety”
(ii) forming hybridization complexes
Para 0023-0025: “(ii) contacting, for each of the one or more target nucleotide sequence, the first probe and the second probe with, preferably for each of the samples in a separate tube, the bridge oligo or plurality of oligonucleotides capable of forming a bridge oligo complex and allow self-annealing into a plurality of ligation complexes…(iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes…(iv) allowing the first target specific portion and the second target specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming a hybridization complex”
(iii) ligating the probes in the one or more hybridization complexes to provide one or more ligated ligation complexes, using a ligase enzyme or enzymes or a combination of a ligase and a DNA polymerase,
Para 0027: “ligating the probes in the hybridization complexes to provide ligated ligation complexes”
Para 0075: “After hybridization, the left and right probe are ligated chemically or enzymatically by a DNA ligase to form ligated ligation complex.”
Para 0179: “FIG. 2B illustrates gap filling between the first probe and the second probe according to an embodiment herein. Here, the bridge oligo contains Gap1 between the bridge sequence 1 (228), and bridge sequence 2 (224). Gap2 is formed between the target binding parts of probes 1 and 2 (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. For this process a mixture of Stoffel fragment, Taq polymerase or Phusion polymerase, and DNA ligase such as Ampligase can be used. The polymerase adds nucleotides (a) complimentary to the universal bridge oligo sequence and (b) complimentary to the target sequence and thereby fills in the two gaps i.e. gap 1 and gap 2 between the first probe and the second probe, and the subsequent action of the DNA ligase results in ligation of the left probe and the right probe complementary to the bridge oligo and the target sequence, into a circular complex.”
(iv) amplifying nucleic acids from the one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase to form an amplified one or more single-stranded concatemeric sequence
Para 0029: “amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase”
(vi) subjecting the one or more amplified single-stranded concatemeric sequence to high-throughput sequencing technology to determine the barcode sequence(s);
Para 0032: “subjecting the concatemeric sequence obtained in step (viii) to high-throughput sequencing technology to determine the barcode sequence(s)”
and (vii) identifying the presence and/or number of the target nucleotide sequence in each of the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode.
Para 0033: “identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target specific portion and/or the second target specific portion, and/or at least part of the first barcode and/or the second barcode, and/or at least part of the third barcode”
Regarding claim 2, Pursiheimo2 disclosed (para [0043]): “…the plurality of samples includes a blood sample, a saliva sample, a urine sample or a feces sample…”.
Regarding claim 6, Pursiheimo2 disclosed (para [0135]-[0138]): “the bridge oligo comprises…(i) one to five 3′ protruding bases, and/or…(ii) 3′ phosphate, and/or…(iii) one or more phosphorothioate modifications within three positions from the 3′ end.”
Regarding claim 7, Pursiheimo2 disclosed (para [0139]): “…wherein the 3′ end of the first probe or the 5′ end of the second probe, or both, are modified to permit chemical ligation of the first probe to the second probe.”
Regarding claim 8, Pursiheimo2 disclosed (para [0140]): “…wherein the bridging portion of the first probe or the second probe, or both, comprise(s) chemically modified bases to permit improved binding to the bridge oligo.”
Regarding claim 9, Pursiheimo2 disclosed (para [0141]): “wherein the first target specific portion, the second target specific portion, the first bridge oligo-specific sequences, and/or the second bridge oligo-specific sequences, contain independently from one another, one or more chemically modified nucleotide.”
Regarding claim 10, Pursiheimo2 disclosed (para [0142]): “…using a phi29 polymerase or a Bst polymerase.”
Regarding claim 11, Pursiheimo2 disclosed (para [0143]): “…wherein a PCR amplification is performed between step (x) and step (xi) using primers which bind to universal parts of the first and second probes, wherein said primers optionally include adapters for subsequent sequencing in step (xi).”
Regarding claim 12, Pursiheimo2 disclosed (para [0144]): “…wherein genetic target enumeration is permitted by counting the number of molecular barcodes per target and per sample.”
Regarding claim 13, Pursiheimo2 disclosed (para [0145]): “…wherein for two or more samples or for two or more locus/allele combinations, barcode sequences are used to genotype the sample(s) for one or more sequences and/or polymorphisms, such as SNPs and/or indels.”
Regarding claim 18, Pursiheimo2 disclosed (para [0028]): “…pooling the ligated ligation complexes from the plurality of samples…”.
Pursiheimo2 did not disclose forming the hybridization complexes as recited in claim 1, whereby the probes were contacted with the target sequence first, followed by contacting that complex with the bridge oligo. Rather, Pursiheimo2 disclosed contacting the probes with the bridge oligo first, followed by contacting that complex with the target sequence.
Also, while Pursiheimo2 disclosed subjecting the concatemeric sequence to sequencing as discussed above, Pursiheimo2 did not disclose
(v-b) bringing the amplified one or more single-stranded concatemeric sequence in contact with a solid support…such that the amplified one or more single-stranded concatemeric sequence become linked to the solid support and separating the solid-support-linked amplified one or more single-stranded concatemeric sequences from components of the samples that are not linked to the solid support…using a solid support capable of binding modified or non-modified DNA with a high affinity
as recited in claim 1, option v-b.
Regarding claim 5, as Pursiheimo2 did not teach capturing the RCA product as recited in step v-b, he did not teach a wash step after step v-b and before step vi (the sequencing step).
Regarding claim 17, Pursiheimo2 disclosed (para [0083]): “A first capture moiety, when used herein, refers to a moiety, such as a chemical group, which allows the probe, ligation complex or hybridization complex to be captured by, i.e. bound to, a second capture moiety which is linked to a solid support.” Pursiheimo2 also disclosed (para [0026]): “optionally, bringing the hybridization complex in contact with a solid support comprising a second capture moiety, allowing the first capture moiety and the second capture moiety to interact such that the hybridization complexes become linked to the solid support and separating the solid-support-linked hybridization complexes from components of the samples that are not linked to the solid-support”. Thus, while Pursiheimo2 disclosed capture of either “ligation complexes” (complexes of the probes and bridging oligo/oligo complex) or “hybridization complexes” (complexes of the probes, the bridging oligo/oligo complex, and target), Pursiheimo2 only taught the “separating” aspect in the case of the “hybridization complexes”, and did not teach capture and separation of the ligated ligation complexes as recited in claim 17.
Larman disclosed an alternative technique in which two probes were first annealed to a target sequence, followed by annealing of a bridge oligo to the probes; see Fig. 2 and para [0034]. While Larman went on to do something else with the resulting product of rolling circle amplification (i.e. annealing detection probes, rather than cleaving into fragments and sequencing), Larman showed that the probes could be annealed to the target first, followed by annealing of a bridge oligo to create the circular template to be used for rolling circle amplification.
Drmanac taught a method for high-throughput sequencing concatemeric products of rolling circle amplification, in which the RCA products (which Drmanac called DNBs—DNA nanoballs) were brought in contact with a solid support, such that the amplified one or more single-stranded concatemeric sequence become linked to the solid support; see Fig. 1, panel C. As discussed in the Drmanac Supplementary Material, the solid support comprised “aminosilane features patterned onto the substrate serve as binding sites for individual DNBs”, and after binding the substrate was “rinsed to neutralize pH and remove unbound DNBs” (pages 6-7, section entitled “DNB array manufacturing”). The term “high affinity” is not defined in the specification as requiring any particular binding strength. Drmanac’s bound DNBs were able to remain bound following rinsing that removed unbound DNBs, and this falls within the broadest reasonable interpretation of “high affinity”.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Pursiheimo2 by contacting the probes to the target sequence first, followed by annealing the bridge oligo to the probes, since Larman showed this to be a workable alternative order of the steps. This difference amounts to combining prior art elements (i.e. particular steps) according to known techniques to yield predictable results.
It would also have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Pursiheimo2 by using Drmanac’s sequencing technique for RCA products, because Drmanac taught this sequencing method “achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs” and offered high accuracy, affordable cost and scalability (abstract). The rejection is not positing replacing Pursiheimo’s entire method with Drmanac’s, but rather only sequencing the RCA products produced by Pursiheimo’s method using the patterned nanoarray technique of Drmanac, which “increased DNA content per array and image information density relative to random genomic DNA arrays” (page 78, third column, lines 12-15). This would also have met the limitations of claim 5, as Drmanac taught a washing (rinsing) between the step of capturing the RCA product on the solid support and the step of sequencing.
Regarding claim 17, as Pursiheimo2 taught capturing and separating “hybridization complexes”, and taught capturing “ligation complexes”, it would have been obvious to perform the separation in the context of “ligation complexes”, as well as capturing and separating “ligated ligation complexes” as recited in claim 17, for the same purpose, i.e. to separate non-productive components of the reaction from productive components.
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pursiheimo (US 2022/0298569, hereinafter “Pursiheimo2”) in view of Larman (US 2023/0039899, previously cited), Drmanac (Science 327:78-81 (2010)) and Drmanac Supplementary Material [online] 2010 [retrieved on 30 October 2025] retrieved from https://www.science.org/doi/full/10.1126/science.1181498 as applied to claims 1, 2, 5-13, 17-18 above, and further in view of Weng (US 2023/0265486).
The teachings of Pursiheimo2, Larman and Drmanac have been discussed. While Drmanac taught capturing and washing the product of the RCA amplification, and Pursiheimo2 disclosed capturing and separating the “hybridization complexes” (para [0026]), none of the references taught capturing and separating the ligated ligation complex (the circularized probes used for the RCA reaction) using a complementary oligonucleotide.
Weng disclosed that in making circularized molecules for sequencing (para [0003]), adaptors could be used (para [0082]), and that circularized molecules comprising the adaptors could be purified away from unligated components using complementary oligonucleotides attached to magnetic beads (para [0083]).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined teachings of Pursiheimo2, Larman and Drmanac by capturing the ligated ligation complexes using complementary oligonucleotides to capture circular ligated molecules since Weng taught this was a way such molecules could be purified away from unligated components.
Response to Arguments
Applicant's arguments filed 02/02/2026 have been fully considered but they are not persuasive.
As the rejections under 35 USC 112(b), Applicant merely points out that the claims have been amended. However, as discussed in the rejection, there are still problems with the claims.
As to the rejections under 35 USC 103, Applicant argues:
PNG
media_image1.png
180
788
media_image1.png
Greyscale
This argument is not persuasive. While Pursiheimo and Larman were performing different overall procedures, both procedures involved the formation of a closed circular nucleic acid molecule by hybridization of probes to both a target and a bridge oligo (the target and bridge oligo serving as scaffolds to place the ends of the probes adjacent one another so that they could be ligated to form the closed circular nucleic acid molecule). In both procedures, the closed circular nucleic acid molecule was used as a template for rolling circle amplification. The teachings of Larman are relevant to those of Pursiheimo in showing that the order in which the components were hybridized prior to ligation did not matter. Therefore, either order would have been obvious.
Next, Applicant argues:
PNG
media_image2.png
204
756
media_image2.png
Greyscale
Pursiheimo does teach pre-assembling the probes and bridge into a ligation complex before sample contact, as discussed in the rejection above, and reiterated here:
PNG
media_image3.png
572
1036
media_image3.png
Greyscale
As to “capture-before-ligation” on a solid support, multi-barcode arrangement for sample-level and molecule-level identification, and pooling of samples after ligation, Applicant is invited to point out any required (not “optional”, not “alternative”) limitation of a rejected claim that is not accounted for in the rejection based on Pursiheimo (US 2022/0298566). Applicant appears to be describing limitations that are either optional, or limitations in claims that were not rejected using this reference.
As to the rejections based on Pursiheimo2 (US 2022/0298569), Applicant argues:
PNG
media_image4.png
112
720
media_image4.png
Greyscale
Applicant is again invited to point to a specific required limitation of a rejected claim for any rejection based either on Pursiheimo or Pursiheimo2. Arguing limitations that are not required by the claims is not persuasive.
Applicant argues:
PNG
media_image5.png
352
766
media_image5.png
Greyscale
This argument is not persuasive. The rejection pointed out that because the combined teachings of Pursiheimo (or Pursiheimo2) and Larman show that either order of hybridizing target, probes, and bridge oligo will allow for ligation of the probes to form a circular template for rolling circle amplification, then either order would have been obvious.
The rejections are therefore maintained.
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 SAMUEL C WOOLWINE whose telephone number is (571)272-1144. The examiner can normally be reached 9am-5:30pm.
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, GARY BENZION can be reached at 571-272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
1 See rationale for obviousness at the end of the rejection for further explanation.
2 Para [0101]-[0105], [0178], and Pursiheimo claim 12.
3 Note that while Pursiheimo’s step designations (i, ii, iii, etc.) may not correspond numerically with the designations for the claimed steps, Pursiheimo’s disclosure teaches the steps of the claimed method when read in context. For example, Pursiheimo’s step viii (para 0029) corresponds functionally to claimed step iv.
4 Note that while Pursiheimo’s step designations (i, ii, iii, etc.) may not correspond numerically with the designations for the claimed steps, Pursiheimo’s disclosure teaches the steps of the claimed method when read in context. For example, Pursiheimo’s step viii (para 0029) corresponds functionally to claimed step iv.
5 Note that while Pursiheimo’s step designations (i, ii, iii, etc.) may not correspond numerically with the designations for the claimed steps, Pursiheimo’s disclosure teaches the steps of the claimed method when read in context. For example, Pursiheimo’s step viii (para 0029) corresponds functionally to claimed step iv.
Prosecution Insights