Methods and devices for characterizing duplex nucleic acid molecules
First Claim
1. A method of characterizing a duplex nucleic acid molecule, said method comprising:
- (a) contacting a conducting fluid medium comprising said duplex nucleic acid with a nanopore;
(b) applying an electric field to said medium and monitoring current changes through said nanopore resulting from said duplex nucleic acid to obtain a set of observed data values; and
(c) characterizing said nucleic acid based on said set of observed data values;
to characterize said duplex nucleic acid molecule.
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Accused Products
Abstract
Methods and devices are provided for characterizing a duplex nucleic acid, e.g., a duplex DNA molecule. In the subject methods, a fluid conducting medium that includes a duplex nucleic acid molecule is contacted with a nanopore under the influence of an applied electric field and the resulting changes in current through the nanopore caused by the duplex nucleic acid molecule are monitored. The observed changes in current through the nanopore are then employed as a set of data values to characterize the duplex nucleic acid, where the set of data values may be employed in raw form or manipulated, e.g., into a current blockade profile. Also provided are nanopore devices for practicing the subject methods, where the subject nanopore devices are characterized by the presence of an algorithm which directs a processing means to employ monitored changes in current through a nanopore to characterize a duplex nucleic acid molecule responsible for the current changes. The subject methods and devices find use in a variety of applications, including, among other applications, the identification of an analyte duplex DNA molecule in a sample, the specific base sequence at a single nulceotide polymorphism (SNP), and the sequencing of duplex DNA molecules.
97 Citations
20 Claims
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1. A method of characterizing a duplex nucleic acid molecule, said method comprising:
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(a) contacting a conducting fluid medium comprising said duplex nucleic acid with a nanopore;
(b) applying an electric field to said medium and monitoring current changes through said nanopore resulting from said duplex nucleic acid to obtain a set of observed data values; and
(c) characterizing said nucleic acid based on said set of observed data values;
to characterize said duplex nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5)
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6. A method of identifying the presence of a duplex nucleic acid molecule in a conducting fluid medium, said method comprising:
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(a) contacting said conducting fluid medium with a nanopore;
(b) applying an electric field to said medium;
(c) monitoring current changes through said nanopore to obtain a set of observed data values; and
(d) determining whether said duplex nucleic acid is present in said conducting fluid medium from said set of observed data values in step (c). - View Dependent Claims (7, 8, 9, 10, 11, 12)
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13. A method of determining the sequence of a duplex DNA molecule, said method comprising:
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(a) providing a fluid conducting medium comprising said duplex DNA molecule as a molecule that is protected at one end and blunt-ended at the other end;
(b) producing a single nucleotide overhang at said blunt end of said duplex DNA molecule;
(c) contacting said fluid conducting medium with a nanopore;
(d) applying an alternating electric field to said fluid conducting medium and monitoring current changes through said nanopore resulting from said duplex nucleic acid to obtain a set of observed data values;
(e) removing said single nucleotide overhang from said duplex DNA molecule;
(f) repeating steps (b) to (e) to obtain a collection of sets of observed data values for each different duplex nucleic acid produced from said original duplex nucleic acid, and (g) determining the sequence of said duplex DNA molecule from said collection of sets of observed data values;
to sequence said duplex DNA molecule. - View Dependent Claims (14)
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15. A nanopore device for characterizing a duplex nucleic acid molecule, said device comprising:
an algorithm for characterizing a duplex nucleic acid molecule based on observed current modulations through a nanopore, wherein said algorithm is present on a computer readable medium.
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16. A kit for use in characterizing a duplex nucleic acid molecule, said kit comprising:
an algorithm for characterizing a duplex nucleic acid molecule based on observed current modulations through a nanopore, wherein said algorithm is present on a computer readable medium.
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17. A kit for use in sequencing a duplex DNA molecule, said kit comprising:
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a first enzyme that produces a single nucleotide overhang comprising duplex DNA molecule from a blunt ended duplex DNA molecule; and
a second enzyme that produce a blunt-ended duplex DNA molecule from a duplex DNA molecule that comprises a single nucleotide overhang. - View Dependent Claims (18, 19, 20)
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Specification