Nanopore-based single DNA molecule characterization, identification and isolation using speed bumps

• US 10,400,278 B2
• Filed: 02/16/2017
• Issued: 09/03/2019
• 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.
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US15/435,206
• Publication Number:
  US 20170159117A1
• Time in Patent Office: 929 Days
• 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 nanopores to obtain sequence information of sample DNAs in ss test DNAs. The method comprises using speed bumps to stall the ss test DNAs in the nanopores at random positions of the ss test DNAs to obtain sequence information of each and every nucleotides of the sample DNAs, and to construct the whole sequences of the sample DNAs. The present invention also relates to identification and/or isolation of test DNAs having desired sequence(s) using nanopore detectors facilitated by speed bump.

Claims

1. A method of detecting one or more identifiers in a single-stranded (ss) test DNA molecule, the method comprising:

(B1) forming a first bulky structure (BS1) on a first end of the ss test DNA molecule, (C1) contacting a pool of speed bumps with the ss test DNA molecule, wherein the speed bumps bind specifically to the one or more identifiers, to form a speed bump-test DNA molecule complex having at least one speed bump-identifier duplex segment,

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

(C3) obtaining a first set of electrical signals when the first speed bump-identifier duplex segment is stalled inside the nanopore for a dwelling time, wherein the first set of electrical signals are characterized to detect the presence of a first identifier,

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

(C5) repeating steps (C1)-(C4) until the ss test DNA molecule is stopped by BS1,

wherein the one or more identifiers are selected from the group consisting of a direction identifier, a reference signal identifier, a sample source identifier, and a sample identifier.

2. The method of claim 1, wherein a first direction identifier is positioned within 20 bases of BS1.

3. The method of claim 2, wherein the ss test DNA molecule further comprises one or more additional identifiers.

4. The method of claim 2, further comprising forming a second bulky structure (BS2) on a second end of the ss DNA molecule.

5. The method of claim 4, wherein a second direction identifier is positioned within 20 bases of BS2.

6. The method of claim 5, wherein the first direction identifier and the second direction identifier are the same.

7. The method of claim 5, wherein the first direction identifier and the second direction identifier are different.

8. The method of claim 5, wherein one or more additional identifiers are positioned between the first direction identifier and the second direction identifier.

9. The method of claim 1, wherein step (C1) forms a speed bump-test DNA molecule complex having at least one speed bump-identifier duplex segment that is up to 16 basepairs.

10. The method of claim 1, wherein the pool of speed bumps is substantially free of speed bumps that bind to non-identifier portions of the ss test DNA.

11. The method of claim 1, wherein the speed bumps that bind specifically to the one or more identifiers and the one or more identifiers each comprise one or more nucleotide analogs selected from the group consisting of iso-deoxyGuanine (isodG), iso-deoxyCytosine (isodC), and combinations thereof.

12. The method of claim 9, wherein sequences of the ssDNA in front of the one or more identifiers in the flow direction of the ss test DNA comprise one or more nucleotide analogs selected from the group consisting of iso-deoxyGuanine (isodG), iso-deoxyCytosine (isodC), and combinations thereof, and/or one or more abasic sites.

13. The method according to claim 1, wherein the ss sample DNA has a length of 5 to 10,000 bases.

14. The method according to claim 1, wherein the nanopore is an alpha hemolysin nanopore detector.

15. The method according to claim 1, wherein the first end is the 5′

end of the ss sample DNA.

16. The method according to claim 1, wherein the first end is the 3′

end of the ss sample DNA.

17. The method of claim 1, further comprising the following steps before step (B1):

(A1) providing a double-stranded (ds) sample DNA,

(A2) ligating a first pre-bulky structure to a first end of the ds sample DNA, and

(A3) denaturing the ds sample DNA of step A2 to the ss test DNA,

wherein step (B1) further comprises forming BS1 from the first pre-bulky structure on the first end of the ss test DNA.

18. The method of claim 15, further comprising forming a second bulky structure (BS2) on a second end of the ss DNA molecule, wherein step (A2) further comprises ligating a second pre-bulky structure to a second end of the ds sample DNA, and wherein step (B1) further comprises forming BS2 from the second pre-bulky structure on the second end of the ss test.