Analyte sequencing with nanopores
AssigneeUniversity of Washington
InventorsJens Gundlach, Ian M. Derrington, Marcus D. Collins
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Abstract
Provided herein are methods and systems pertaining to sequencing units of analytes using nanopores. In general, arresting constructs are used to modify an analyte such that the modified analyte pauses in the opening of a nanopore. During such a pause, an ion current level is obtained that corresponds to a unit of the analyte. After altering the modified analyte such that the modified analyte advances through the opening, another arresting construct again pauses the analyte, allowing for a second ion current level to be obtained that represents a second unit of the analyte. This process may be repeated until each unit of the analyte is sequenced. Systems for performing such methods are also disclosed.
Claims19
A method of detecting two or more nucleotides of an analyte comprising a nucleic acid, the method comprising:
- (a) providing a nanopore positioned between a cis side comprising a first conductive liquid medium and a trans side comprising a second conductive liquid medium, wherein the nanopore provides liquid communication between the cis side and the trans side, wherein the analyte is present on the cis side, the analyte comprising a first arresting construct;
- (b) causing the analyte that comprises the first arresting construct to advance into the nanopore, whereby the first arresting construct pauses the advance of the analyte toward the trans side, thereby producing a first residual ion current level that represents a first nucleotide or set of nucleotides adjacent to the first arresting construct, wherein the first residual ion current level is different than a measurable ion current of the same nucleotide or set of nucleotides in an analyte that is not arrested;
- (c) altering the first arresting construct of the analyte, the alteration allowing the analyte to advance toward the trans side, thereby changing the first residual ion current level produced in (b);
- (d) causing a second arresting construct to pause the advance of the analyte toward the trans side, thereby changing the ion current level produced in (c);
- and
- (e) detecting the changes in the ion current levels to distinguish the first nucleotide or set of nucleotides from a second nucleotide.
A method of detecting two or more nucleotides of a nucleic acid, comprising:
- (a) providing a nucleic acid comprising at least two nucleotides or set of repeat nucleotides each defined as Xn, wherein n=1-1,000,000 and wherein each X and each n may be the same or different;
- (b) placing a first insert arresting construct in the nucleic acid, the first insert arresting construct comprising a duplex nucleic acid and being adjacent to an Xn, to provide a modified nucleic acid;
- (c) providing a nanopore positioned between a cis side comprising a first conductive liquid medium and the modified nucleic acid and a trans side comprising a second conductive liquid medium;
- (d) causing the first insert arresting construct to pause the modified nucleic acid upon entering the nanopore from the cis side, thereby producing a first residual ion current level that represents the Xn adjacent to the first arresting construct, wherein the first residual ion current level is different than a measurable ion current of the same Xn in an analyte that is not arrested;
- (e) altering the first insert arresting construct, the alteration allowing the nucleic acid to advance toward the trans side, thereby changing the first residual ion current level produced in (d);
- (f) causing a second insert arresting construct to pause the nucleic acid upon entering the nanopore, thereby changing the ion current level produced in (e);
- and
- (g) comparing the changes in the ion current levels to distinguish two or more nucleotides of the nucleic acid.
A method of correlating an ion current level with a known nucleotide unit of an analyte comprising a nucleic acid, the method comprising:
- (a) providing a nanopore positioned between a cis side comprising a first conductive liquid medium and an analyte modified with a first arresting construct and containing all known nucleotide units of interest, and a trans side comprising a second conductive liquid medium, wherein the nanopore provides liquid communication between the cis side and the trans side;
- (b) causing the first arresting construct to pause the advance of the analyte toward the trans side, thereby producing a first residual ion current level representing a first known nucleotide unit of the analyte that is adjacent to the first arresting construct, wherein the first residual ion current level is different than the measurable ion current of the same nucleotide in an analyte that is not arrested;
- (c) altering the first arresting construct of the modified analyte, the alteration allowing the modified analyte to advance toward the trans side;
- (d) causing a second arresting construct to pause the advance of the analyte toward the trans side, thereby producing a second residual ion current level representing a second known nucleotide unit of the analyte;
- and
- (e) calibrating the nanopore with a known analyte containing all nucleotide units and corresponding ion current levels of interest and optionally obtaining a separator level, thereby correlating each ion current level with a known unit of an analyte.
Dependencies are inferred from the claim text.