Nanopore-based nucleic acid analysis with mixed FRET detection

• US 9,862,997 B2
• Filed: 05/23/2014
• Issued: 01/09/2018
• Est. Priority Date: 05/24/2013
• Status: Active Grant

• Current Assignee: Quantapore, Inc.
• Original Assignee: Quantapore, Inc.
• Inventors: Huber, Martin, Clancy, Bason E., Hardenbol, Paul
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US14/786,518
• Publication Number:
  US 20160076091A1
• Time in Patent Office: 1,327 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

Various methods, systems and devices for optical detection and analysis of polymers, such as polynucleotides, using nanopores, e.g., for determining sequences of nucleic acids, are provided herein. In certain variations, methods and systems for determining a nucleotide sequence of a polynucleotide, which include measuring mixed FRET signals as a polynucleotide translocates through a nanopore and determining a nucleotide sequence of the polynucleotide from the mixed FRET signals, are provided.

Claims

1. A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:

translocating a polynucleotide through a nanopore so that nucleotides of the polynucleotide pass in sequence by a FRET donor positioned adjacent to the nanopore, a plurality of the nucleotides being within a FRET distance of the FRET donor as the nucleotides exit the nanopore and at least a portion of the nucleotides being labeled with a FRET acceptor, the nanopore dimensioned so that acceptor labels on the nucleotides inside the nanopore are constrained to suppress FRET reactions and wherein different kinds of nucleotides are labeled with FRET acceptors that generate distinguishable signals;

exciting the FRET donor adjacent to the nanopore so that FRET occurs between the FRET donor and a plurality of FRET acceptors on the plurality of nucleotides emerging from the nanopore and within the FRET distance to generate a mixed FRET signal;

measuring the mixed FRET signals within the FRET distance as the polynucleotide translocates through the nanopore;

separating the distinguishable signals from the mixed FRET signal of each measurement; and

determining a nucleotide sequence of the polynucleotide from the measured distinguishable signals separated from the mixed FRET signals collected from every nucleotide of the polynucleotide.

2. The method of claim 1, wherein said nanopore is disposed in a solid phase membrane and wherein said FRET donor is attached to the solid phase membrane adjacent to said nanopore.

3. The method of claim 1, wherein said nanopore is a protein nanopore and wherein said FRET donor is attached to the protein nanopore.

4. The method of claim 1, wherein the polynucleotide is a single stranded polynucleotide.

5. The method of claim 1, wherein said step of exciting said FRET donor includes exposing said FRET donor to an illumination beam.

6. A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:

translocating a polynucleotide through a nanopore having an exit so that nucleotides of the polynucleotide pass in sequence through a FRET zone upon exiting the nanopore, the FRET zone encompassing a plurality of the nucleotides during such passage and at least a portion of the nucleotides being labeled with FRET acceptors and at least one FRET donor being in the FRET zone, the nanopore dimensioned so that acceptor labels on the nucleotides inside the nanopore are constrained to suppress FRET reactions and wherein different kinds of nucleotides are labeled with FRET acceptors that generate distinguishable signals;

exciting the FRET donor in the FRET zone so that FRET occurs between the FRET donor and a plurality of FRET acceptors on the plurality of nucleotides emerging from the nanopore and in the FRET zone to generate a mixed FRET signal;

measuring the mixed FRET signals within the FRET zone as the polynucleotide moves through the FRET zone;

separating the distinguishable signals from the mixed FRET signal of each measurement; and

determining a nucleotide sequence of the polynucleotide from the measured distinguishable signals separated from the mixed FRET signals collected from every nucleotide of the polynucleotide.

7. The method of claim 6, wherein said nanopore is disposed in a solid phase membrane and wherein said FRET donor is attached to the solid phase membrane adjacent to said nanopore.

8. The method of claim 6, wherein said nanopore is a protein nanopore and wherein said FRET donor is attached to the protein nanopore.

9. The method of claim 6, wherein the polynucleotide is a single stranded polynucleotide.

10. The method of claim 6, wherein said step of exciting said FRET donor includes exposing said FRET donor to an illumination beam.

11. A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:

translocating a polynucleotide with acceptor-labeled nucleotides through a nanopore dimensioned so that acceptor labels on the nucleotides inside the nanopore are constrained to suppress FRET reactions, wherein nucleotides of the polynucleotide pass in sequence through a FRET zone of a FRET donor upon exiting the nanopore, wherein the FRET zone encompasses a plurality of the nucleotides during such passage, and wherein different kinds of nucleotides are labeled with acceptors that generate distinguishable signals;

exciting the FRET donor in the FRET zone so that FRET occurs between the plurality of acceptor-labeled nucleotides in the FRET zone and the FRET donor to generate a mixed FRET signal;

measuring the mixed FRET signals within the FRET zone as the polynucleotide moves through the FRET zone;

separating the distinguishable signals from the mixed FRET signal of each measurement; and

determining a nucleotide sequence of the polynucleotide from the measured distinguishable signals separated from the mixed FRET signals collected from every nucleotide of the polynucleotide.

12. The method of claim 11, wherein said nanopore is disposed in a solid phase membrane and wherein FRET donor is attached to the solid phase membrane adjacent to said nanopore.

13. The method of claim 11, wherein said nanopore is a protein nanopore and wherein said FRET donor is attached to the protein nanopore.

14. The method of claim 11, wherein the polynucleotide is a single stranded polynucleotide.

15. The method of claim 11, wherein at least one of said acceptors is a fluorescent organic dye.

16. The method of claim 11, wherein said FRET donor is a quantum dot.

17. The method of claim 11, wherein said step of exciting said FRET donor includes exposing said FRET donor to an illumination beam.