Coupling method

Patent metadata

Field	Value
Publication number	US-10246741-B2
Application number	US-201214122573-A
Country	US
Kind code	B2
Filing date	May 25, 2012
Priority date	May 27, 2011
Publication date	Apr 2, 2019
Grant date	Apr 2, 2019

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 for detecting polynucleotides, comprising:
(a) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(b) contacting the membrane, in an ionic solution, with polynucleotides, wherein following contact with the membrane the polynucleotides are tethered to the membrane via a tethering group;
(c) applying a potential difference across the membrane and detecting a first polynucleotide using the nanopore, from among the polynucleotides tethered to the membrane; and
(d) detecting a second polynucleotide, from among the polynucleotides tethered to the membrane, using the same nanopore, wherein the second polynucleotide is not the first polynucleotide.

2. The method according to claim 1 , wherein the membrane is an amphiphilic layer or a solid state layer.

3. The method according to claim 1 , wherein the membrane is a lipid bilayer.

4. The method according to claim 1 , wherein the tethering group comprises a hydrophobic anchor.

5. The method according to claim 4 , wherein the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube or amino acid.

6. The method according to claim 4 , wherein the hydrophobic anchor is capable of embedding in the membrane.

7. The method according to claim 1 , wherein the tethering group comprises a linker.

8. The method according to claim 7 , wherein the linker comprises a polynucleotide, polyethylene glycol, polysaccharide, or polypeptide.

9. The method according to claim 1 , wherein the polynucleotide is tethered transiently to the membrane.

10. The method according to claim 1 , wherein the first polynucleotide and the second polynucleotide are detected based on ion flow through the nanopore that is measured via an electrical means.

11. The method according to claim 1 , wherein the nanopore is a protein nanopore.

12. The method according to claim 11 , wherein the protein nanopore is derived from Msp or α-hemolysin (α-HL).

13. The method according to claim 1 , wherein the nanopore comprises a molecular adaptor that mediates interaction of the polynucleotide with the nanopore.

14. The method according to claim 1 , wherein the nanopore is coupled to a polynucleotide binding protein, which is optionally an exonuclease or a polymerase.

15. The method according to claim 1 , wherein the first polynucleotide and the second polynucleotide are detected based on ion flow through the nanopore that is measured by measuring a current passing through the nanopore.

16. The method according to claim 1 , wherein the polynucleotide comprises a target polynucleotide.

17. The method according to claim 16 , wherein the method comprises digesting a target polynucleotide to provide a fragment and the fragment sequence is determined.

18. The method according to claim 1 , wherein the tethering group comprises a polypeptide.

19. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of less than 0.01 pM.

20. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of less than 0.1 pM.

21. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of less than 1 pM.

22. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of less than 10 pM.

23. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of less than 100 pM.

24. The method according to claim 1 , wherein the polynucleotide is present in the solution of (b) at a concentration of about 0.001 pM to about 1 nM.

25. The method according to claim 1 , wherein rate of interaction of the molecules of the polynucleotide with the nanopore is increased as compared to rate of interaction of molecules of the polynucleotide with the nanopore in the absence of the tethering group.

26. The method according to claim 25 , wherein the rate of interaction of the molecules of the polynucleotide is increased by at least three orders of magnitude.

27. The method according to claim 1 , wherein the effective concentration of the polynucleotide at the nanopore is increased as compared to the concentration of the polynucleotide in the solution of (b).

28. The method according to claim 1 , wherein a single nanopore providing an ion channel through the membrane is present in the membrane.

29. The method according to claim 1 , wherein the polynucleotide is functionalized with the tethering group.

Assignees

• Clarke James
• White James
• Milton John
• Brown Clive
• Oxford Nanopore Tech Ltd

Inventors

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

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · 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

• G01N27/44743

  Introducing samples · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title