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.
Claims21
A system comprising:
- a protein nanopore positioned between a cis side comprising a first conductive liquid medium and a trans side comprising a second conductive liquid medium, wherein the protein nanopore is disposed in an artificial membrane spanning the opening of an aperture, wherein the membrane comprises a mycolic acid and has a rupture voltage greater than 1.0 V when voltage applied across the membrane is ramped at about 100 mV/s in the presence of a 1.0 M KCl solution prepared with deionized water, and buffered to pH 8.0±0.05 with 10 mM HEPES, and wherein the protein nanopore provides liquid communication between the cis side and the trans side, and
- a data acquisition device operable to detect an ion current through the protein nanopore;
- a nucleic acid analyte in the first liquid medium, wherein the nucleic acid analyte comprises a first arresting construct and a second arresting construct, and wherein the first arresting construct is configured to pause the advance of the nucleic acid analyte toward the trans side after entry of the nucleic acid analyte into the protein nanopore for a time sufficient to detect a first ion current level and then allow the nucleic acid analyte to advance toward the trans side.
A system comprising:
- a protein nanopore positioned between a cis side comprising a first conductive liquid medium and a trans side comprising a second conductive liquid medium, wherein the protein nanopore is disposed in an artificial membrane spanning the opening of an aperture, wherein the membrane comprises a mycolic acid and has a rupture voltage greater than 1.0 V when voltage applied across the membrane is ramped at about 100 mV/s in the presence of a 1.0 M KCl solution prepared with deionized water, buffered to pH 8.0±0.05 with 10 mM HEPES and a voltage greater than 1.0 V is sustained for greater than 1 hour, and wherein the protein nanopore provides liquid communication between the cis side and the trans side, and
- a data acquisition device operable to detect an ion current through the protein nanopore;
- a nucleic acid analyte in the first liquid medium, wherein the nucleic acid analyte comprises a first arresting construct and a second arresting construct, wherein the first arresting construct is configured to pause the advance of the nucleic acid analyte toward the trans side after entry of the nucleic acid analyte into the protein nanopore for a time sufficient to detect a first ion current level and then allow the nucleic acid analyte to advance toward the trans side, wherein the second arresting construct is configured to pause the advance of the nucleic acid analyte toward the trans side after the first arresting construct has allowed the nucleic acid analyte to advance for a time sufficient to detect a second ion current level, and wherein one of the first arresting construct and the second arresting construct is positioned on the nucleic acid analyte such that the first ion current level or the second ion current level is a separator level that differs from any nucleic acid unit-specific ion current level.
Dependencies are inferred from the claim text.