Analysis of a polynucleotide via a nanopore system

Patent metadata

Field	Value
Publication number	US-12351867-B2
Application number	US-202117369834-A
Country	US
Kind code	B2
Filing date	Jul 7, 2021
Priority date	Dec 19, 2012
Publication date	Jul 8, 2025
Grant date	Jul 8, 2025

Abstract

A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.

First claim

The invention claimed is:

1. A method of analyzing a target polynucleotide that comprises at each nucleotide position a member of a set of different nucleotides, the method comprising:
A) obtaining an expanded single-stranded polynucleotide that comprises a sequence of expanded units, wherein each expanded unit:
a) ordinally corresponds to a particular nucleotide position of the target polynucleotide, and
b) comprises:
i) a clock nucleotide sequence, the clock nucleotide sequence being common among expanded units of the expanded polynucleotide, and ii) at least one signal nucleotide, the at least one signal nucleotide being indicative of the member of the set of different nucleotides at the particular nucleotide position;

B) hybridising two or more single-stranded polynucleotides to the expanded single-stranded polynucleotide to form an expanded polynucleotide comprising two or more sections of double-stranded polynucleotide separated by single-stranded polynucleotide;
C) translocating the expanded polynucleotide through a nanopore until a first section of double-stranded polynucleotide reaches the nanopore and halts translocation of the expanded polynucleotide;
D) making electrical measurements dependent on the expanded polynucleotide within the pore;
E) un-hybridising the first section of double-stranded polynucleotide such that translocation of the expanded polynucleotide through the nanopore continues; and
F) determining the sequence of one or more regions of the target polynucleotide based on the measurements obtained in step D).

2. A method according to claim 1 , wherein steps C to E are repeated with respect to a second and subsequent sections of double-stranded polynucleotide.

3. A method according to claim 1 , wherein step B comprises hybridising a single-stranded polynucleotide to each expanded unit present in the single-stranded polynucleotide.

4. A method according to claim 1 , wherein determining the sequence of the one or more regions of the target polynucleotide comprises deriving an analysis signal from the measurements obtained in step D.

5. A method according to claim 4 , wherein determining the sequence of the one or more regions of the target polynucleotide further comprises analyzing the analysis signal by:
identifying, within the analysis signal, clock signals, wherein each clock signal is derived from at least one of the clock nucleotides in the clock nucleotide sequence in an expanded unit ordinally corresponding to the clock signal; and
identifying nucleotide signals, wherein each nucleotide signal is derived from the at least one signal nucleotide in an expanded unit ordinally corresponding to the nucleotide signal; and
analyzing the nucleotide signals to analyze the target polynucleotide.

6. A method according to claim 5 , wherein the nucleotide signal derived from the at least one signal nucleotide is indicative of the member of the set of different nucleotides at the particular nucleotide position of the target polynucleotide.

7. A method according to claim 5 , wherein the step of analyzing the nucleotide signals comprises analyzing the nucleotide signals to determine the sequence of the target polynucleotide or one or more regions thereof.

8. A method according to claim 7 , wherein the step of analyzing the nucleotide signals to determine the sequence of the target polynucleotide or one or more regions thereof comprises deriving, from the nucleotide signals, a feature vector of time-ordered features representing characteristics of the measurements and determining similarity between the derived feature vector and at least one other feature vector.

9. A method according to claim 5 , wherein the step of analyzing the nucleotide signals comprises analyzing the nucleotide signals to determine the identity of individual nucleotides in the target polynucleotide.

10. A method according to claim 4 , wherein the step of deriving an analysis signal comprises detecting states in electrical measurements, and deriving, from each state, at least one value representing a characteristic of the state to form said analysis signal.

11. A method according to claim 1 , wherein the value of each measurement is dependent upon a k-mer, being a group of k nucleotide units where k is greater than one.

12. A method according to claim 11 , where k is 7 or greater.

13. A method according to claim 1 , wherein the clock nucleotide sequence comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the clock nucleotide sequence.

14. A method according to claim 1 , wherein the at least one signal nucleotide consists of a single signal nucleotide.

15. A method according to claim 14 , wherein the single signal nucleotide has the same identity as the member of the set of different nucleotides at the particular nucleotide position.

16. A method according to claim 1 , wherein the at least one signal nucleotide consists of a signal nucleotide sequence of plural signal nucleotides, wherein the signal nucleotide sequence is indicative of the member of the set of different nucleotides at the particular nucleotide position.

17. A method according to claim 16 , wherein one of the plural signal nucleotides has the same identity as the member of the set of different nucleotides at the particular nucleotide position.

18. A method according to claim 16 , wherein the clock nucleotide sequence and the signal nucleotide sequence are in a predetermined order in which the signal nucleotide sequence is contiguous.

19. A method according to claim 1 , wherein the electrical measurements comprise measurements of ion flow through the nanopore.

20. A method according to claim 1 , wherein the nanopore is a biological pore.

21. A method according to claim 1 , wherein said translocation of the polynucleotide through the nanopore is performed in a ratcheted manner in which successive nucleotides are registered with the nanopore.

22. A method according to claim 1 , further comprising, before said step of making electrical measurements, expanding the target polynucleotide to form the expanded single-stranded polynucleotide.

23. A method according to claim 1 , wherein at least one signal nucleotide of the at least one signal nucleotides has the same identity as the member of the set of different nucleotides at the particular nucleotide position.

24. A method according to claim 1 , wherein at least one signal nucleotide of the at least one signal nucleotides is the complement of the member of the set of different nucleotides at the particular nucleotide position.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Reid Stuart William
• Harper Gavin

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title