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.
Claims24
A method of sequencing two or more units of an analyte modified with at least a first arresting construct and a second arresting construct, comprising:
- (a) providing a nanopore positioned between a cis side comprising a first conductive liquid medium and the modified analyte 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 of the modified analyte to pause the modified analyte upon entering the nanopore, thereby producing a first ion current level representing the first unit;
- (c) altering the first arresting construct of the modified analyte, the alteration allowing the modified analyte to advance toward the trans side;
- and
- (d) causing the second arresting construct of the modified analyte to pause the modified analyte upon entering the nanopore, thereby producing a second ion current level representing the second unit.
A method of sequencing two or more nucleotides of a nucleic acid, comprising:
- (a) providing a nucleic acid comprising at least two unknown nucleotides or set of unknown 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 and a second insert arresting construct in the nucleic acid, each comprising a duplex nucleic acid and each 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 a first insert arresting construct to pause the modified nucleic acid upon entering the nanopore from the cis side, thereby producing a first ion current level, wherein the first ion current level represents a first Xn;
- (e) altering the first insert arresting construct, the alteration allowing the modified nucleic acid to advance toward the trans side;
- (f) causing the second insert arresting construct of the modified nucleic acid to pause upon entering the nanopore, thereby producing a second ion current level representing a second Xn;
- and
- (g) comparing the first ion current level and the second ion current level with an ion current level of a known Xn, thereby sequencing two or more nucleotides of the nucleic acid.
A method of correlating an ion current level with a known unit of an analyte, comprising:
- (a) providing a nanopore positioned between a cis side comprising a first conductive liquid medium and an analyte modified with two or more arresting constructs and containing all known 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 a first arresting construct of the modified analyte to pause the modified analyte upon entering the nanopore from the cis side, thereby producing an ion current level representing a first known unit of the analyte;
- (c) altering the first arresting construct of the modified analyte, the alteration allowing the modified analyte to advance toward the trans side;
- (d) causing the second arresting construct of the modified analyte to pause the modified analyte upon entering the nanopore, thereby producing a second ion current level representing the second unit;
- and
- (e) calibrating the nanopore with a known modified analyte containing all 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.
A system comprising a nanopore, wherein the nanopore is positioned between a first conductive liquid medium and a second conductive liquid medium, and wherein at least one liquid medium comprises a modified nucleic acid having first and second arresting constructs.
Dependencies are inferred from the claim text.