Selective modification of polymer subunits to improve nanopore-based analysis

• US 10,017,814 B2
• Filed: 09/02/2014
• Issued: 07/10/2018
• Est. Priority Date: 08/30/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated, University of Washington
• Original Assignee: Illumina Incorporated, University of Washington
• Inventors: Gundlach, Jens H., Laszlo, Andrew, Derrington, Ian, Mandell, Jeffrey G.
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US14/915,611
• Publication Number:
  US 20160222444A1
• Time in Patent Office: 1,407 Days
• Field of Search: None
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The present disclosure provides method and systems for improving nanopore-based analysis of polymers. The disclosure provides methods for selectively modifying one or more monomeric subunit(s) of a kind a pre-analyte polymer that results polymer analyte with a modified subunit. The polymer analyte produces a detectable signal in a nanopore-based system. The detectable signal, and/or its deviation from a reference signal, indicates the location of the modified subunit in the polymer analyte and, thus, permits the identification of the subunit at that location in the original pre-analyte polymer.

Claims

1. A method for analyzing a polymer analyte, comprising:

(a) selectively modifying a target polymer subunit of a kind in a pre-analyte polymer to produce a polymer analyte with an abasic site, wherein selectively modifying the target polymer subunit of a kind comprises contacting the target polymer subunit of a kind with an agent to produce a modified subunit and converting the modified subunit to an abasic site;

(b) translocating the polymer analyte comprising the abasic site from a first conductive liquid medium to a second conductive liquid medium through a nanopore, wherein the nanopore provides liquid communication between the first conductive liquid medium and the second conductive liquid medium;

(c) measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as the polymer analyte passes through the nanopore; and

(d) detecting the abasic site based on the measured ion current.

2. The method of claim 1, wherein step (a) further comprises selectively modifying a plurality of a target polymer subunits of the same kind in the pre-analyte polymer.

3. The method of claim 2, wherein the pre-analyte polymer comprises a nucleic acid, a DNA, an RNA;

a PNA;

a polypeptide;

or a combination thereof.

4. The method of claim 3, wherein the kind of target polymer subunit is a cytosine residue, a guanine residue, a thymine residue, an adenine residue, or a uracil residue.

5. The method of claim 4, wherein selectively modifying the cytosine residue comprises selectively converting the cytosine residue into a uracil residue.

6. The method of claim 5, wherein converting the cytosine residue into the uracil residue comprises contacting the pre-analyte polymer with an agent selected from bisulfite, cytosine deaminase, NO, N₂O₃, and Echinomycin.

7. The method of claim 5, wherein selectively modifying the cytosine residue further comprises converting the uracil residue into an abasic site, thereby producing the polymer analyte.

8. The method of claim 7, wherein converting the uracil residue into an abasic site comprises contacting the pre-analyte polymer with a nucleic acid error correction enzyme.

9. The method of claim 8, wherein the nucleic acid error correction enzyme is uracil deglycosylase (UNG).

10. The method of claim 4, wherein either the kind of target polymer subunit is a cytosine residue and selectively modifying the cytosine residue comprises selectively methylating the cytosine residue, or the kind of polymer subunit is a guanine residue and selectively modifying the guanine residue comprises selectively methylating the guanine residue.

11. The method of claim 10, wherein selectively methylating the cytosine residue comprises contacting the pre-analyte polymer with a methyltransferase enzyme.

12. The method of claim 10, wherein selectively modifying the cytosine residue or guanine residue further comprises converting the methylated cytosine residue into an abasic site, thereby producing the polymer analyte.

13. The method of claim 2, wherein step (d) comprises:

(i) comparing the measured ion current to an ion current corresponding to a reference polymer comprising the same sequence as the analyte polymer wherein the subunit of a kind in the reference polymer is not modified; and

(ii) detecting the presence or absence of a difference in the ion currents compared in step (i), wherein the presence or absence of a difference in ion currents indicates the presence or absence of the subunit modification in the polymer analyte, respectively.

14. The method of claim 13, further comprising translocating the reference polymer from the first conductive liquid medium to the second conductive liquid medium through the nanopore and measuring an ion current to provide the ion current corresponding to the reference polymer.

15. The method of claim 1, further comprising determining the position of the abasic site in the polymer analyte based on a characteristic of the measured ion current.

16. The method of claim 15, comprising determining the identity of the target polymer subunit at a position in the pre-analyte polymer sequence that corresponds to the position of the modified subunit in the polymer analyte.

17. The method of claim 2, further comprising:

performing the steps (a) through (c) for a plurality of pre-analyte polymers that comprise a common sequence;

producing a consensus map of the plurality of ion currents measured in step (c); and

detecting the presence of multiple modified subunits in the common sequence.

18. A method for analyzing a nucleic acid analyte, comprising:

(a) incorporating a modified nucleobase into the nucleic acid analyte;

(b) contacting the nucleic acid analyte with an error correction enzyme capable of removing the modified nucleobase to provide an abasic site in the nucleic acid analyte;

(c) translocating the nucleic acid analyte from a first conductive liquid medium to a second conductive liquid medium through a nanopore, wherein the nanopore provides liquid communication between the first conductive liquid medium and the second conductive liquid medium;

(d) measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as the nucleic acid analyte passes through the nanopore; and

(e) detecting the abasic site based on the measured ion current.