US 12,352,742 B2
Methods and compositions for generating reference maps for nanopore-based polymer analysis
Jens Gundlach, Seattle, WA (US); Ian M. Derrington, Seattle, WA (US); Andrew Laszlo, Seattle, WA (US); and Elizabeth Manrao, Seattle, WA (US)
Assigned to University of Washington through its Center for Commercialization, Seattle, WA (US)
Filed by University of Washington through its Center for Commercialization, Seattle, WA (US)
Filed on Oct. 12, 2023, as Appl. No. 18/486,103.
Application 18/486,103 is a continuation of application No. 16/824,186, filed on Mar. 19, 2020, granted, now 11,959,133.
Application 16/824,186 is a continuation of application No. 14/395,793, granted, now 10,612,083, issued on Apr. 7, 2020, previously published as PCT/US2013/037454, filed on Apr. 19, 2013.
Claims priority of provisional application 61/635,768, filed on Apr. 19, 2012.
Prior Publication US 2024/0158846 A1, May 16, 2024
Int. Cl. G01N 33/487 (2006.01); C12Q 1/6869 (2018.01); G01N 27/447 (2006.01)
CPC G01N 33/48721 (2013.01) [C12Q 1/6869 (2013.01); G01N 27/447 (2013.01)] 19 Claims
OG exemplary drawing
 
1. A method for determining characteristics of an unknown analyte polymer in a nanopore system, wherein the nanopore system has a multi-subunit output signal resolution and comprises a nanopore positioned in a nonconductive barrier between a first conductive liquid medium and a second conductive liquid medium that are in liquid communication through the nanopore, wherein the multi-subunit output signal resolution is determined by a multi-subunit combination, and wherein the multi-subunit combination comprises a plurality of contiguous subunits of polymer subunits in a constriction region of the nanopore at any given time, the method comprising:
(i) applying voltage across the nonconductive barrier;
(ii) translocating the unknown analyte polymer through the nanopore of the nanopore system; and
(iii) detecting an ion current output signal;
(iv) generating a reference map in the nanopore system, the method comprising:
(a) applying a voltage across the nonconductive barrier;
(b) translocating one or more reference polymers through the nanopore of the nanopore system to generate a plurality of reference ion current output signals, wherein the one or more reference polymers comprise the same type of polymer as the unknown polymer, and wherein the one or more reference polymers comprise a reference polymer sequence such that each possible multi-subunit combination that can determine an output signal according to the output resolution of the nanopore system appears only once in the reference polymer sequence; and
(c) compiling the plurality of reference output signals into a reference map, wherein the reference map is suitable for sequence analysis of the unknown polymer in the nanopore system; and
(v) comparing the ion current level in (iii) to the reference map to provide unknown analyte polymer sequence information.