Modified base detection with nanopore sequencing
First Claim
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1. A method for sequencing a nucleic acid template and identifying modified bases therein 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;
controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid;
measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation;
determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and
determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme from the interaction of the modified base with the processive enzyme.
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Abstract
Methods, compositions, and systems are provided for characterization of modified nucleic acids. Nanopore sequencing is performed using a processive enzyme to control the rate of translation of a single strand of a nucleic acid through a nanopore. The presence and identity of a modified base can be determined by monitoring the kinetics of translation through the pore even though the events that lead to the kinetic changes are separated in time and space from the translation of the modified base or it'"'"'s compliment through the nanopore. The invention also comprises the use of hemi-genomic DNA in nanopore sequencing.
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Citations
20 Claims
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1. A method for sequencing a nucleic acid template and identifying modified bases therein 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; controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid; measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation; determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme from the interaction of the modified base with the processive enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 16, 17, 18, 19, 20)
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7. A method for sequencing a nucleic acid 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; controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid; measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation; determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme due to the interaction of the modified base with the processive enzyme; wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15)
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Specification