Analyte sequencing with nanopores
AssigneeUniversity of Washington
InventorsJens H. 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.
Claims20
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;
- (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 that represents a second nucleotide or set of nucleotides adjacent to the second arresting construct;
- and
- (e) detecting a difference between the first residual ion current level and the second residual ion current level to distinguish the first nucleotide or set of nucleotides from a second nucleotide or set of nucleotides.
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;
- (d) causing a second arresting construct to pause the advance of the analyte toward the trans side, thereby producing a second residual ion current that represents a second nucleotide or set of nucleotides adjacent to the second arresting construct;
- and
- (e) comparing the first residual ion current level and/or the second residual ion current level with one or more ion current levels of known nucleotides, thereby identifying nucleotides of the analyte.
A method of detecting two or more nucleotides of a nucleic acid, comprising:
- (a) 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;
- (b) providing a nucleic acid in the first conductive liquid medium, wherein the nucleic acid comprises 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, and wherein the nucleic acid comprises a first pendant arresting construct that comprises a duplex nucleic acid and is adjacent to an Xn;
- (c) causing the first pendant arresting construct to pause the 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 pendant 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;
- (d) altering the first pendant arresting construct, the alteration allowing the nucleic acid to advance toward the trans side;
- (e) causing a second pendant arresting construct to pause the nucleic acid upon entering the nanopore, thereby producing a second residual ion current level;
- and
- (f) comparing the first residual ion current level and the second residual ion current level to distinguish two or more nucleotides of the nucleic acid.
Dependencies are inferred from the claim text.