NANOPORE-BASED SINGLE DNA MOLECULE CHARACTERIZATION USING SPEED BUMPS

• US 20120160688A1
• Filed: 12/21/2011
• Published: 06/28/2012
• Est. Priority Date: 12/22/2010
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Davis, Randall W., Chen, Roger J.A.
• Granted Patent: US 9,121,059 B2
• Time in Patent Office: Days
• US Class Current: 204/543
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• 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
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

The present invention relates to a method of using nanopore to obtain sequence information of an unknown structure (unknown DNA) in a ss test DNA. The method comprises using speed bump to stall the ss test DNA in the nanopore at random positions of the ss test DNA to obtain sequence information of each and every nucleotides of the unknown DNA, and to construct the whole sequence of the unknown DNA. The present invention also relates to a novel method of trapping a ss test DNA in a nanopore using two bulky structures formed under different conditions (e.g. different temperature), and the bulky structures are able to keep the ss test DNA trapped in a nanopore at a working temperature.

Claims

1. A method of obtaining sequence information of an unknown sequence in a ss test DNA, comprising:

(B1) forming a first bulky structure (BS1) on a first end of the ss test DNA at a first temperature,

(B2) applying a first electric potential to flow the ss test DNA through a nanopore,

(B3) forming a second bulky structure (BS2) on a second end of the ss test DNA at a second temperature,

(B4) optionally applying another electric potential to reverse the flow of the ss test DNA until the ss test DNA is stopped by BS2 before the constriction area of the nanopore,

(B5) contacting a random speed bump pool with the ss test DNA to form a speed bump-test DNA complex having at least one speed bump-test DNA duplex segment at a working temperature,

(B6) applying a third electric potential to flow the speed bump-test DNA complex through the nanopore until a first speed bump-test DNA duplex segment is stopped before the constriction area of the nanopore,

(B7) obtaining a first set of electrical signals when the first speed bump-test DNA duplex segment is stalled inside the nanopore for a dwelling time, and characterizing the nucleotide sequence that is in front of the first speed bump-test DNA duplex segment and the first basepair of the first speed bump-test DNA duplex segment, in the flow direction of the ss test DNA,

(B8) dissociating the first speed bump-test DNA duplex segment and continuing the flow of the DNA through the nanopore,

(B9) repeating steps (B4)˜

(B8) until the ss test DNA is stopped by BS1 or BS2,

(B10) applying another electric potential to move the test DNA at a reversed direction of the test DNA flow in step (B5) until the ss test DNA is stopped by the other bulky structure before the constriction area of the nanopore,

(B11) repeating steps (B9)˜

(B10) at least 1 time, and

(B12) constructing the ss unknown DNA sequence by overlapping the collected nucleotide sequence information,

2. The method according to claim 1, wherein the first temperature is higher than the second temperature, which is higher than the working temperature.

3. The method according to claim 2, wherein the working temperature is at about −

10 to about 20°

C.

4. The method according to claim 2, wherein the working temperature is about −

10 to about 15°

C.

5. The method according to claim 2, wherein the speed bumps have a length of up to 8 bases.

6. The method according to claim 2, wherein the speed bumps have a length of up to 6 bases.

7. The method according to claim 2, wherein the speed bumps have a length of up to 4 bases.

8. The method according to claim 2, wherein PB2 has a sequence of SEQ ID NO. 1.

9. The method according to claim 2, wherein in step (B11), steps (B9)˜

(B10) are repeated at least 10 times.

10. The method according to claim 2, wherein in step (B11), steps (B9)˜

(B10) are repeated at least 20 times.

11. The method according to claim 2, wherein the ss unknown DNA has a length of 5 to about 10,000 bases.

12. The method according to claim 2, wherein the ss unknown DNA has a length of 15 to about 10,000 bases.

13. The method according to claim 2, wherein the nanopore is an alpha hemolysin nanopore detector.

14. The method according to claim 2, wherein the amount of the electric potentials applied in steps (B4)˜

(B11) are the same or different, or are continuously changing.

15. The method according to claim 1, wherein the random speed bump pool comprises speed bumps having universal base at 3′

end and/or 5′

end.

16. The method according to claim 1, wherein the random speed bump pool comprises speed bumps which form duplex sections with ss test DNA, and the duplex sections have about the same melting temperatures.

17. The method according to claim 1, wherein the random speed bump pool comprises speed bumps having universal bases.

18. The method according to claim 1, wherein the random speed bump pool comprises speed bumps composed of locked nucleotides.

19. The method according to claim 1, wherein the random speed bump pool comprises speed bumps composed of locked nucleotides having universal bases.

20. The method according to claim 1, wherein the random speed bump pool comprises speed bumps having dideoxynucleosides at the 3′

end and/or the 5′

end.

21. The method according to claim 1, wherein the random speed bump pool comprises speed bump trains formed by linking multiple speed bumps with non-binding linkers.

22. The method according to claim 1, wherein BS1 and/or BS2 comprise(s) structures that are non-binding to the random speed bump pool.

23. The method according to claim 22, wherein the non-binding structures are selected from the group consisting of isodG, isodC, abasic site and combination thereof.

24. A method of obtaining sequence information of a single-stranded (ss) test DNA having an identifier segment having a known sequence, comprising:

(B1) forming a first bulky structure on a first end of the test DNA molecule,

(C1) contacting a pool of speed bumps (speed bump pool) with the test DNA molecule to form a speed bump-test DNA molecule complex having at least one speed bump-test DNA molecule segment,

(C2) applying an electric potential to flow the speed bump-test DNA molecule complex through a nanopore until a first speed bump-test DNA molecule segment is stalled before the constriction area of the nanopore,

(C3) obtaining a first set of electrical signals when the first speed bump-test DNA molecule segment is stalled inside the nanopore for a dwelling time, in the flow direction of the test DNA molecule,

(C4) dissociating the first speed bump-test DNA molecule segment and continuing the flow of the molecule through the nanopore, and

(C5) repeating steps (C1)˜

(C4) until the test DNA molecule is stopped by BS1 and

(C6) according to the first set of electrical signals obtained in step (C3) and/or the electrical signals obtained from step (C5),

indicating the ss test DNA comprises the identifier segment, and/or

characterizing the nucleotide sequence that is in front of the speed bump-identifier duplex segment and the nucleotide sequence of the first basepair of the speed bump-test DNA duplex segment, in the flow direction of the ss test DNA.

25. The method according to claim 24, wherein the nucleotide sequence that is in front of the first speed bump-test DNA duplex segment is a 1, 2, 3, 4, or 5-base sequence.

26. The method according to claim 25, further comprising the following steps:

(B2) applying a second electric potential to flow the ss test DNA through a nanopore, and

(B3) forming a second bulky structure (BS2) on a second end of the ss test DNA at a second temperature, wherein;

the first temperature is at least 10°

C. higher than the second temperature, and the second temperature is at least 5°

C. higher than the working temperature.