Coupling method

Patent metadata

Field	Value
Publication number	US-12152276-B2
Application number	US-202017094571-A
Country	US
Kind code	B2
Filing date	Nov 10, 2020
Priority date	May 27, 2011
Publication date	Nov 26, 2024
Grant date	Nov 26, 2024

Abstract

The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.

First claim

The invention claimed is:

1. A method of preparing a double-stranded target polynucleotide for nanopore sequencing, the method comprising:
(a) obtaining a target polynucleotide;
(b) hybridizing a primer with a single stranded leader to the target polynucleotide, extending the primer using a polymerase to copy the target polynucleotide;
(c) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(d) applying a potential difference across the membrane, wherein the polynucleotide is captured by the nanopore via the leader; and
(e) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the nanopore.

2. The method of claim 1 , wherein (b) is repeated by polymerase chain reaction (PCR).

3. The method of claim 1 , wherein the primer comprises a complementary section to the target polynucleotide and a homopolymer section.

4. The method of claim 3 , wherein the primer prevents the polymerase from extending beyond the homopolymer section.

5. The method of claim 4 , wherein the leader comprises a single-stranded polyT section.

6. The method of claim 1 , wherein the membrane is an amphiphilic layer, a lipid bilayer, or a solid state layer.

7. The method of claim 1 , further comprises contacting the membrane with the target polynucleotide, wherein the target polynucleotide is tethered to the membrane via an anchor.

8. The method of claim 7 , wherein the anchor is a hydrophobic anchor selected from a group consisting of a lipid, a fatty acid, a sterol, a carbon nanotube, and an amino acid.

9. The method of claim 7 , wherein the hydrophobic anchor is capable of embedding in the membrane.

10. The method of claim 7 , wherein the polynucleotide is tethered transiently to the membrane.

11. The method of claim 1 , wherein the target polynucleotide is detected based on ion flow through the nanopore that is measured via an electrical means.

12. The method of claim 1 , wherein the polynucleotide is captured by the nanopore via the leader by a DNA handling protein.

13. The method of claim 12 , wherein the DNA handling protein is an exonuclease or a polymerase.

14. The method of claim 1 , wherein the polynucleotide is detected based on ion flow through the nanopore by measuring a current passing through the nanopore.

15. The method of claim 1 , wherein the target polynucleotide is a fragment of genomic DNA.

16. A method for strand sequencing of a double-stranded target polynucleotide, the method comprising:
(a) obtaining a target polynucleotide;
(b) hybridizing a primer with a single stranded leader to the target polynucleotide, extending the primer using a polymerase to copy the target polynucleotide;
(c) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(d) applying a potential difference across the membrane, wherein the polynucleotide is captured by the nanopore via the leader; and
(e) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the nanopore.

17. The method of claim 16 , further comprises contacting the membrane with the target polynucleotide, wherein the target polynucleotide is tethered to the membrane via an anchor.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Clarke James Anthony
• White James
• Milton John
• Brown Clive Gavin

Classifications

• G01N27/44743

  Introducing samples · CPC title

• G01N33/48721

  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) · CPC title

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• G01N33/487

  of liquid biological material · CPC title