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
Applicant’s amendment filed 11/26/2025 is acknowledged. Regarding the Office action mailed 08/27/2025, the rejection of claim 7 under 35 USC 102(a)(1) is moot in view of the cancellation of claim 7.
Upon further review, new grounds of rejection are set forth below. This Office action is NON-FINAL.
Claim Interpretation
Claim 1 recites: “A method for determining type of a nucleotide on a nucleic acid sequence to be analyzed…”. Neither of these terms is explicitly defined in the specification. The word “determining” could mean “elucidating”, but it could also mean “choosing”. One could “elucidate” what nucleotide is present at a given position in a nucleic acid to be analyzed. One could also “choose” what nucleotide is present in at a give position in a nucleic acid being synthesized. This leads to the meaning of “to be analyzed”. The reference WO 2019/150134 discloses a method for synthesizing a nucleic acid molecule using a nanopore. However, the reference also discloses that, once synthesized, “the final polynucleotide synthesis molecule can be characterised on the same nanopore by nanopore sequencing”. Therefore, the nucleic acid molecule being synthesized was a nucleic acid molecule “to be analyzed”.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Heron (WO 2019/150134 A1, IDS reference).
Regarding claim 1, Heron disclosed:
A method for determining type of a nucleotide on a nucleic acid sequence
Page 2, lines 21-28. By incorporating a desired nucleotide at a given position in a polynucleotide being synthesized, Heron is “determining” the type of nucleotide in the nucleic acid sequence.
to be analyzed,
Page 124, line 5: “At the end of the synthesis cycles, the final polynucleotide synthesis molecule can be characterised on the same nanopore by nanopore sequencing.” Thus, the polynucleotide being synthesized was a nucleic acid “to be analyzed”.
comprising the steps of: (a) providing at least one nucleotide molecule in a first compartment and a nucleic acid sequence to be analyzed in a second compartment, with the first compartment and the second compartment separated by a membrane having at least one nanopore;
Page 2, lines 21-28. The “polynucleotide synthesis molecule” on the trans side of the nanopore is the nucleic acid sequence “to be analyzed” in a second compartment. The “transfer nucleotide” on the cis side of the nanopore is the “nucleotide molecule” in a first compartment. See page 3, lines 1-3, where Heron discloses that the nucleotide is attached to a “feeder molecule” (see, e.g., Fig. 4, 7).
(b) applying an electric field to drive the nucleotide molecule and/or a part thereof to pass through the nanopore in a first direction or to be inserted in the nanopore;
Page 2, lines 23-24: “…moving the transfer nucleotide through the channel of a nanopore disposed in a substrate from the cis side to the trans side of the substrate…”.
Page 3, line 17: “…moving the feeder molecule through the nanopore to bring the attached transfer nucleotide into contact with the enzyme whereupon the enzyme catalyses the transfer of the transfer nucleotide to the polynucleotide synthesis molecule…”
Page 71, lines 8-30; e.g. “A feeder molecule comprising the transfer nucleotide or a feeder molecule without the transfer nucleotide would be typically charged and drawn into the nanopore under a potential difference depending upon the polarity of the electrodes and the charge of the molecule being drawn into the nanopore.”
(c) measuring a first current characteristic value passing through the membrane in the state step of (b) to identify the nucleotide molecule and/or a part thereof;
Page 4, lines 11-12: “A first verification process may be performed to determine the identity and/or integrity of the transfer nucleotide…”.
Page 6, lines 1-2: “…the first verification process may be performed whilst the feeder molecule is at least partially within the channel of the nanopore.”
Page 7, lines 15-19: “Measurement of the feeder molecule or the transfer nucleotide may be made with respect to the nanopore. Preferably, the feeder molecule or the transfer nucleotide is measured by measuring ion current flow through the nanopore under the action of a potential difference applied across the substrate.”
Page 79, lines 14-18: “As discussed herein, a determination of the identity of a polymer unit may be achieved by measuring a property with respect to the nanopore as the unit passes through the nanopore. In particular, such determination may be achieved by measuring the extent to which a polymer unit alters or perturbs the ionic current flowing through the nanopore under the action of an applied potential.”
(d) applying an electric field in a direction opposite to that of the electric field applied in step (b) or in the same direction but with a lower driving voltage, causing the nucleotide molecule and/or a part thereof to pass through the nanopore and/or exit the nanopore in a second direction, with the second direction being opposite to the first direction;
Page 3, lines 23-25: “…moving the feeder molecule through the nanopore to the cis…side of the substrate following transfer of the transfer nucleotide to the polynucleotide synthesis molecule.”
Page 71, lines 8-30; e.g. “Movement of the portion of the feeder molecule back through the nanopore in the trans to cis direction can be achieved by the action of a reverse applied potential, e.g. a reverse voltage bias, applied across the substrate.”
(e) measuring a second current characteristic value passing through the membrane to identify the nucleotide molecule and/or a part thereof,
Page 6, line 29 through page 7, line 5: “In any of the polynucleotide extension methods described and defined herein involving feeder molecules, step (C) may be performed only following a second verification process performed to verify that the enzyme has catalysed the transfer of the transfer nucleotide from the feeder molecule to the polynucleotide synthesis molecule, wherein the second verification process comprises:
I. moving the feeder molecule through the nanopore in the cis direction, and determining the presence or absence of the nucleobase of the transfer nucleotide…”.1
Page 7, lines 15-19: “Measurement of the feeder molecule or the transfer nucleotide may be made with respect to the nanopore. Preferably, the feeder molecule or the transfer nucleotide is measured by measuring ion current flow through the nanopore under the action of a potential difference applied across the substrate.”
comparing the second current characteristic value with a pre-determined standard current characteristic value of the nucleotide molecule or polyphosphate molecule in the state of step (d) to determine whether the nucleotide molecule and/or a part thereof is attached to the nucleic acid sequence to be analyzed, and thus determining the type of nucleotide on the nucleic acid sequence to be analyzed in the second compartment.
Page 83, line 30 through page 84, line 11: “The second verification process may comprise a measurement comparison performed in conjunction with a first verification process. In such a comparison a first verification process, as described above, is performed to establish the identity, and optionally the integrity, of the transfer nucleotide whilst moving the feeder molecule through the nanopore in the cis direction before the transfer nucleotide is contacted with the enzyme, thus obtaining a first measurement corresponding to the desired transfer nucleotide when attached to the feeder molecule. A second measurement of the same feeder molecule is obtained during the step of determining the presence or absence of the nucleobase of the attached transfer nucleotide whilst moving the feeder molecule back through the nanopore in the cis direction. The first and second measurements are then compared to determine a change in the measurements which may provide an indication that the transfer nucleotide has been removed from the feeder molecule, i.e. by the action of the enzyme.”
Regarding claim 2, see figure 7A. See also page 9, lines 11-17: “In any of the polynucleotide extension methods described and defined herein, the transfer nucleotide may be provided as a molecule comprising a nucleobase, a ribose sugar and three or more phosphate groups.
In any of the polynucleotide extension methods described and defined herein, the nucleotide may be a deoxy nucleotide triphosphate (dNTP), such as dATP, dTTP, dCTP or dGTP, or a modified dNTP such as a modified dATP, a modified dTTP, a modified dCTP and/or a modified dGTP.”
See also page 10, lines 23-29: “In any of the polynucleotide extension methods described and defined herein, a transfer nucleotide may be attached to a feeder molecule via a linker. In any such method the linker may comprise a phosphate group linker comprising one phosphate group, or a polyphosphate group linker comprising two, three or more phosphate groups. The linker may comprise a hydrocarbon chain, e.g. comprising from 2 to 20 or more carbon atoms, optionally comprising an alkylene group e.g. a C2-20 alkylene group.”
See also page 11, lines 9-20; e.g. “…wherein a blocking moiety is a peptide, oligopeptide, polypeptide, protein or other polymer.”
Regarding claim 3, see page 66, line 29 through page 67, line 2: “A biological nanopore may be provided in a biological substrate. The biological nanopore is inserted into the biological substrate. A biological substrate may be a membrane, such as an amphiphilic layer, for example a lipid bilayer.”
Regarding claim 4, see page 64, line 13: “The biological pore may be a naturally occurring pore or may be a mutant pore.” See also page 63, line 11: “A biological nanopore may be a transmembrane protein pore.”
Regarding claim 5, see page 32, lines 5-14: “A polynucleotide synthesis molecule comprising a double-stranded polynucleotide molecule or portion is provided in proximity to an enzyme on the trans side of the substrate.
The enzyme is any suitable enzyme capable of extending a polynucleotide synthesis molecule, wherein the polynucleotide synthesis molecule is one strand of a double-stranded molecule comprising nucleic acid. In order to extend a polynucleotide synthesis molecule, an enzyme, for example a polymerase, may require the polynucleotide synthesis molecule to be hybridised to another nucleic acid molecule. In this way, the polynucleotide synthesis molecule acts as a primer for the enzyme, and the primer is extended by the normal function of the enzyme.”
Alternatively, see page 103, lines 15-18: “For example in methods for the synthesis of single stranded polynucleotide synthesis molecule, the polynucleotide synthesis molecule may be converted into a double stranded molecule by the action of a further polymerase and primer using the single stranded polynucleotide synthesis molecule as a complementary template.”
Regarding claim 6, see page 103, lines 9-13: “In any of the methods defined herein, the extension process may be a first extension process and wherein the methods further comprise repeating the extension process one or more times to further extend the polynucleotide synthesis molecule with one or more further transfer nucleotides to synthesise a polynucleotide having a predefined sequence.” See also page 124, lines 5-6: “At the end of the synthesis cycles, the final polynucleotide synthesis molecule can be characterised on the same nanopore by nanopore sequencing.”
Conclusion
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.
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/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681
1 Step C, as discussed on page 3, lines 23-25, is: “moving the feeder molecule through the nanopore to the cis or trans side of the substrate following transfer of the transfer nucleotide to the polynucleotide synthesis molecule.”