Analyte sequencing with nanopores

摘要

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.

主权项

1. 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.