Nanopore sequencing methods
First Claim
1. A method for sequencing a nucleic acid and determining the presence and position of a modified base in a template nucleic acid using a nanopore comprising:
- providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;
providing a voltage across the nanopore to produce a measurable current flow through the nanopore;
providing, in the upper solution, a polymerase-nucleic acid complex comprising a strand displacing polymerase and a circular nucleic acid template which may have modified bases and providing the components required for nucleic acid synthesis, whereby the polymerase produces a nascent strand that passes through the nanopore as the nascent strand is produced;
measuring the current through the nanopore over time as the nascent strand is translated through the nanopore;
determining a sequence of the nascent strand as it translates through the nanopore using the measured current over time, and determining the presence of a modified base in the circular nucleic acid template using a measured change in kinetics of the polymerase, whereby the polymerase proceeds around the circular template, then continues to produce nascent strand such that a sequence in the nascent strand and the presence of a modified base in the circular template is determined more than once, anddetermining the position of the modified base using a determined value for the number of bases between the active site and the bases within the nanopore.
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Abstract
Methods are provided for sequencing of nucleic acid templates using nanopores. A polymerase-nucleic acid complex having a strand displacing polymerase is provided. The nucleic acid in the polymerase-nucleic acid complex is a circular nucleic acid template. Components required for nucleic acid synthesis are provided whereby the polymerase produces a nascent strand that passes through the nanopore while the nascent strand is produced. Current through the nanopore over time is measured allowing for the determination of the sequence of the nascent strand as it translates through the pore using the measured current over time. Because the polymerase can proceed around the circular template multiple times, multiple reads corresponding to the same sequence in the circular template can be obtained in one sequencing process. Where modified bases are present in the template, the method can be used to determine the presence and position of the modified bases using the kinetics of the polymerase.
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Citations
20 Claims
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1. A method for sequencing a nucleic acid and determining the presence and position of a modified base in a template nucleic acid using a nanopore comprising:
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providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid; providing a voltage across the nanopore to produce a measurable current flow through the nanopore; providing, in the upper solution, a polymerase-nucleic acid complex comprising a strand displacing polymerase and a circular nucleic acid template which may have modified bases and providing the components required for nucleic acid synthesis, whereby the polymerase produces a nascent strand that passes through the nanopore as the nascent strand is produced; measuring the current through the nanopore over time as the nascent strand is translated through the nanopore; determining a sequence of the nascent strand as it translates through the nanopore using the measured current over time, and determining the presence of a modified base in the circular nucleic acid template using a measured change in kinetics of the polymerase, whereby the polymerase proceeds around the circular template, then continues to produce nascent strand such that a sequence in the nascent strand and the presence of a modified base in the circular template is determined more than once, and determining the position of the modified base using a determined value for the number of bases between the active site and the bases within the nanopore. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification