Analyte sequencing with nanopores
Inventors
Gundlach, Jens • Derrington, Ian M. • Collins, Marcus D.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9588079-B2
Publication Date
2017-03-07
Expiration Date
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.
Core Innovation
The invention provides a nanopore positioned between a cis side and a trans side, where a nucleic-acid analyte on the cis side includes a first arresting construct. The analyte advances into the nanopore so that the first arresting construct pauses advance toward the trans side, producing a first residual ion current level that represents a first nucleotide or set of nucleotides adjacent to the first arresting construct.
The first arresting construct is altered to allow the analyte to advance toward the trans side, thereby changing the residual ion current level produced in the paused state. A second arresting construct then pauses advance of the analyte toward the trans side, producing an altered ion current level, and changes in ion current levels are detected to distinguish nucleotides.
In related embodiments, a first insert arresting construct comprising a duplex nucleic acid is placed adjacent to X^n repeat nucleotides to provide a modified nucleic acid that is paused in the nanopore to generate residual ion current levels representing X^n adjacent to the first insert arresting construct. The second insert arresting construct can provide a separator level that is distinct from any nucleotide-specific ion current level, and ion-current changes are compared to distinguish two or more nucleotides.
Claims Coverage
Three independent claims are provided: clm-00001, clm-00017, and clm-00019. Across these claims, the independent inventive features center on sequential pausing using arresting constructs, generating residual ion current levels to represent nucleotide units adjacent to arresting constructs, altering arresting constructs to change residual ion current levels, and correlating or distinguishing nucleotide identities by comparison and calibration.
Residual ion current discrimination using sequential arresting constructs
Providing a nanopore between a cis side and a trans side with an analyte on the cis side that comprises a first arresting construct; causing the analyte to advance into the nanopore so the first arresting construct pauses advance to produce a first residual ion current level representing a first nucleotide or set of nucleotides adjacent to the first arresting construct; altering the first arresting construct to change the first residual ion current level; causing a second arresting construct to pause advance to change the ion current level produced; and detecting changes in the ion current levels to distinguish the first nucleotide or set of nucleotides from a second nucleotide.
Repeat-defined insert arresting constructs for distinguishing nucleotides
Providing a nucleic acid comprising at least two nucleotides or set of repeat nucleotides each defined as X^n, placing a first insert arresting construct comprising a duplex nucleic acid adjacent to X^n to provide a modified nucleic acid, providing a nanopore positioned between a cis side and the modified nucleic acid and a trans side, causing the first insert arresting construct to pause the modified nucleic acid in the nanopore to produce a first residual ion current level representing the X^n adjacent to the first insert arresting construct, altering the first insert arresting construct to change the first residual ion current level, causing a second insert arresting construct to pause the nucleic acid to change the ion current level produced, and comparing the changes in the ion current levels to distinguish two or more nucleotides of the nucleic acid.
Calibration-based correlation of residual ion current levels with known nucleotide units
Providing a nanopore between a cis side and a trans side, where the nanopore receives an analyte modified with a first arresting construct and containing all known nucleotide units of interest; causing the first arresting construct to pause advance to produce a first residual ion current level representing a first known nucleotide unit adjacent to the first arresting construct; altering the first arresting construct to allow advance; causing a second arresting construct to pause advance to produce a second residual ion current level representing a second known nucleotide unit; and calibrating the nanopore with a known analyte containing all nucleotide units and corresponding ion current levels of interest and optionally obtaining a separator level to correlate each ion current level with a known unit of an analyte.
The independent claims collectively cover detecting or distinguishing nucleotides by pausing a modified nucleic-acid analyte at nucleotide-adjacent positions using arresting constructs, altering those constructs to change residual ion current levels, and using ion-current changes, and optionally a distinct separator level, to distinguish nucleotide identities. They also include correlating observed residual ion current levels with known nucleotide units through nanopore calibration using a known analyte containing all nucleotide units of interest.
Stated Advantages
Produces residual ion current levels that represent nucleotide units adjacent to arresting constructs, where the residual ion current level is different than a measurable ion current of the same nucleotide in an analyte that is not arrested.
Enables distinguishing nucleotides by detecting changes in ion current levels produced by arresting and altering arresting constructs.
Enables correlating ion current levels with known nucleotide units by calibrating the nanopore with a known analyte containing all nucleotide units of interest.
Optionally provides a separator level distinct from any nucleotide-specific ion current level to support distinguishing nucleotide identities.
Documented Applications
Detecting two or more nucleotides of an analyte comprising a nucleic acid, including distinguishing a first nucleotide or set of nucleotides from a second nucleotide using changes in ion current levels.
Detecting nucleotides of a nucleic acid that includes at least two nucleotides or set of repeat nucleotides defined as X^n by using insert arresting constructs comprising duplex nucleic acids and comparing ion-current changes.
Correlating an ion current level with a known nucleotide unit of an analyte by calibrating the nanopore with a known analyte containing all nucleotide units of interest and corresponding ion current levels, optionally obtaining a separator level.
Interested in licensing this patent?