HAIRPIN LOOP METHOD FOR DOUBLE STRAND POLYNUCLEOTIDE SEQUENCING USING TRANSMEMBRANE PORES

• US 20150152492A1
• Filed: 07/25/2012
• Published: 06/04/2015
• 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, Heron, Andrew John
• Granted Patent: US 9,957,560 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 double stranded target polynucleotide, comprising:

(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety;

(b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;

(c) moving the single stranded polynucleotide through a transmembrane pore such that a proportion of the nucleotides in the single stranded polynucleotide interact with the pore; and

(d) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target polynucleotide,

wherein the separating in step (b) comprises contacting the construct with a polynucleotide binding protein which separates the two strands of the target polynucleotide.

2. The method according to claim 1, wherein the bridging moiety comprises a polymeric linker, a chemical linker, a polynucleotide or a polypeptide.

3. The method according to claim 2, wherein the bridging moiety comprises DNA, RNA, modified DNA or RNA, PNA, LNA or PEG.

4. The method according to claim 2, wherein the bridging moiety is a hairpin loop.

5. The method according to claim 1, wherein the entire single stranded polynucleotide moves through the pore and the entire target polynucleotide is sequenced.

6. The method according to claim 1, wherein a polynucleotide binding protein controls the movement of the single stranded polynucleotide though the transmembrane pore.

7. The method according to claim 1, wherein the polynucleotide binding protein separates the two strands of the target polynucleotide and controls the movement of the single stranded polynucleotide through the pore.

8. The method according to claim 1, wherein the protein is:

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

(b) derived from Phi29 DNA polymerase;

or

(c) derived from a helicase.

9. The method according to claim 1, wherein the construct further comprises at least one polymer at the opposite end of the target polynucleotide to the bridging moiety.

10. The method according to claim 9, wherein the construct comprises a polymer leader on the one strand of the target polynucleotide and a polymer tail on the other strand of the target polynucleotide.

11. The method according to claim 10, wherein the single stranded polynucleotide moves through the pore in the order of (1) the leader polymer, (2) the one strand of the target polynucleotide, (3) the bridging moiety, (4) the other strand of the target polynucleotide and (5) the tail polymer.

12. The method according to claim 1, wherein the construct further comprises means of coupling the construct to the membrane.

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

14. The method according to claim 13, wherein the protein pore is derived from Msp or α

-hemolysin (α

-HL).

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

16. The method according to claim 15, wherein the membrane is a lipid bilayer.

17. The method according to claim 1, wherein the construct further comprises one or more markers which result in a distinctive current when they interact with the pore.

18. The method according to claim 17, wherein the one or more markers are abasic or a specific sequence of nucleotides.

19. The method according to claim 17, wherein the one or more markers are in or near the bridging moiety.

20. The method according to claim 17, wherein the one or more markers are positioned in the polymer leader or the polymer tail.

21. The method according to claim 17, wherein the one or more markers identify the source of the target polynucleotide.

32. An apparatus for sequencing a double stranded target polynucleotide, comprising:

(a) a membrane;

(b) a plurality of transmembrane pores in the membrane;

(c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide; and

(d) instructions for carrying out the method of claim 1.

33. An apparatus for sequencing a double stranded target polynucleotide, comprising:

(a) a membrane;

(b) a plurality of transmembrane pores in the membrane; and

(c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide, wherein the apparatus is set up to carry out the method of claim 1.

34. An apparatus according to claim 32, wherein the apparatus comprises:

a sensor device that is capable of supporting the membrane and plurality of pores and being operable to perform polynucleotide sequencing using the pores and proteins;

at least one reservoir for holding material for performing the sequencing;

a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and

a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device.

35. A method according to claim 1, further comprising optionally after step (b) and before step (c):

synthesising a complement of the single stranded polynucleotide, such that the single stranded polynucleotide and complement form a double stranded polynucleotide;

linking the two strands of the double stranded polynucleotide at or near one end of the double stranded polynucleotide using a bridging moiety;

separating the two strands of the double stranded polynucleotide to provide a further single stranded polynucleotide comprising the original single stranded polynucleotide linked to the complement by the bridging moiety.

22. A kit for preparing a double stranded target polynucleotide for sequencing comprising:

(a) a bridging moiety capable of linking the two strands of the target polynucleotide at or near one end; and

(b) at least one polymer.

23. The kit according to claim 22, comprising a leader polymer and a tail polymer.

24. The kit according to claim 23, wherein the leader polymer and tail polymer are polynucleotides and a portion of the leader polymer and a portion of the tail polymer form a double stranded sequence.

25. The kit according to claim 22, further comprising means of coupling the target polynucleotide to a membrane.

26. The kit according to claim 22, further comprising one or more markers which result in a distinctive current when they interact with a transmembrane pore.

27. A method of preparing a double stranded target polynucleotide for sequencing, comprising:

(a) linking the two strands of the target polynucleotide at or near one end with a bridging moiety; and

(b) attaching one polymer to one strand at the other end of the target polynucleotide and thereby forming a construct that allows the target polynucleotide to be sequenced using a transmembrane pore.

28. The method according to claim 27, wherein the polymer is a leader polymer and the method further comprises attaching a tail polymer to the other strand of the target polynucleotide at the same end as the leader polymer.

29. The method according to claim 28, further comprising attaching to the construct means to couple the construct to a membrane.

30. The method according to claim 27 wherein:

(a) the bridging moiety is synthesised separately and is chemically attached or enzymatically ligated to the target polynucleotide;

or

(b) the bridging moiety is generated in the processing of the target polynucleotide.

31. A method of sequencing a double stranded target polynucleotide, comprising:

(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety;

(b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;

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

(d) linking the two strands of the double stranded polynucleotide at or near one end of the double stranded polynucleotide using a bridging moiety;

(e) separating the two strands of the double stranded polynucleotide to provide a further single stranded polynucleotide comprising the original single stranded polynucleotide linked to the complement by the bridging moiety;

(f) moving the complement through a transmembrane pore such that a proportion of the nucleotides in the complement interact with the pore; and

(g) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target polynucleotide,

wherein the separating in step (e) comprises contacting the construct with a polynucleotide binding protein which separates the two strands of the target polynucleotide.