Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

• US 9,957,560 B2
• Filed: 07/25/2012
• Issued: 05/01/2018
• Est. Priority Date: 07/25/2011
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Brown, Clive Gavin, Clarke, James Anthony, Hall, Graham, Harper, Gavin, Heron, Andrew John, White, James
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/234,698
• Publication Number:
  US 20150152492A1
• Time in Patent Office: 2,106 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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 invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

Claims

1. A method of sequencing a target polynucleotide using an apparatus comprising:

(i) a chamber comprising an aqueous solution and a membrane separating the chamber into two compartments, wherein a transmembrane nanopore is present in the membrane; and

(ii) electrodes connected to the two compartments, the method comprising;

(a) providing in the solution (i) a construct comprising a double stranded, target polynucleotide, wherein a single stranded leader polynucleotide is linked to one end of the double stranded, target polynucleotide, and a hairpin loop links the two strands of the target polynucleotide at the opposite end of the target polynucleotide; and

(ii) a polynucleotide binding protein that is a polymerase or a helicase that binds the construct;

(b) applying a voltage across the electrodes sufficient to cause the construct to move, in a single stranded form, through the nanopore, wherein under the applied voltage the polynucleotide binding protein simultaneously controls the rate of movement of the target polynucleotide through the nanopore and separates the two strands of the target polynucleotide to generate the single stranded form; and

(c) measuring the current passing through the nanopore as the construct moves through the nanopore in the order of (1) one strand of the target polynucleotide, (2) the hairpin loop, and (3) the other strand of the target polynucleotide, and sequencing the target polynucleotide based on the measured current.

2. The method according to claim 1, wherein the hairpin loop comprises a polynucleotide or a polypeptide.

3. The method according to claim 2, wherein the hairpin loop comprises DNA, RNA, abasic DNA or a basic RNA, PNA, LNA or PEG.

4. The method according to claim 1, wherein the entire construct moves, in the single stranded form, through the nanopore and the entire target polynucleotide is sequenced.

5. The method according to claim 1, wherein the polynucleotide binding protein is:

(a) a polymerase from a virus of the Picovirinae family;

(b) a Phi29 DNA polymerase;

or

(c) a DNA helicase.

6. The method according to claim 1, wherein the target polynucleotide comprises a polynucleotide leader on the one strand of the target polynucleotide and a polynucleotide tail on the other strand of the target polynucleotide.

7. The method according to claim 6, wherein the construct moves through the nanopore in the order of (1) the polynucleotide leader, (2) the one strand of the target polynucleotide, (3) the hairpin loop, (4) the other strand of the target polynucleotide and (5) the polynucleotide tail.

8. The method according to claim 1, wherein the target polynucleotide further comprises a means of coupling the construct to the membrane.

9. The method according to claim 1, wherein the transmembrane nanopore is a protein nanopore or a solid state nanopore.

10. The method according to claim 9, wherein the protein nanopore is derived from Msp or α

-hemolysin (α

-HL).

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

12. The method according to claim 11, wherein the membrane is a lipid bilayer.

13. The method according to claim 1, wherein the construct further comprises one or more markers which result in a distinctive current when they pass through the nanopore, wherein the one or more markers comprises abasic DNA or abasic RNA, a nucleotide sequence for differentiating between the two strands of the target polynucleotide, a nucleotide with an unnatural base, a fluorophore or cholesterol.

14. The method according to claim 13, wherein the one or more markers are in the hairpin loop.

15. The method according to claim 13, wherein the one or more markers are positioned in the polynucleotide leader on one strand of the target polynucleotide or a polynucleotide tail on the other stand of the target polynucleotide.

16. A method of sequencing a target polynucleotide using an apparatus comprising:

(i) a chamber comprising an aqueous solution and a membrane separating the chamber into two compartments, wherein a transmembrane nanopore is present in the membrane; and

(ii) electrodes connected to the two compartments, the method comprising;

(a) providing a single stranded polynucleotide comprising two strands of a double stranded, target polynucleotide, wherein the two strands of the target polynucleotide are linked at one end of the target polynucleotide by a polynucleotide linker;

(b) synthesising a complement of the single stranded polynucleotide of step (a), such that the single stranded polynucleotide of step (a) and the complement form a double stranded polynucleotide;

(c) providing in the solution (i) a construct comprising the double stranded polynucleotide of step (b), wherein a single stranded leader polynucleotide is linked to one end of the double stranded polynucleotide and a hairpin loop links the two strands of the double stranded polynucleotide at the opposite end of the double stranded polynucleotide; and

(ii) a polynucleotide binding protein that is a polymerase or a helicase that binds the construct;

(d) applying a voltage across the electrodes sufficient to cause the construct to move, in a single stranded form, through the nanopore, wherein under the applied voltage the polynucleotide binding protein simultaneously controls the rate of movement of the double stranded polynucleotide of step (b) through the transmembrane nanopore and separates the two strands of the double stranded polynucleotide of step (b) to generate the single stranded form; and

(e) measuring the current passing through the nanopore as the construct moves through the nanopore in the order of (1) the single stranded polynucleotide of step (a), (2) the hairpin loop, and (3) the complement of step (b), and sequencing the target polynucleotide based on the measured current.