ULTRAFAST SEQUENCING OF BIOLOGICAL POLYMERS USING A LABELED NANOPORE

• US 20120261261A1
• Filed: 03/21/2012
• Published: 10/18/2012
• Est. Priority Date: 09/30/2009
• Status: Active Grant

• Current Assignee: Quantapore, Inc.
• Original Assignee: Quantapore, Inc.
• Inventors: HUBER, Martin
• Granted Patent: US 8,771,491 B2
• Time in Patent Office: Days
• US Class Current: 204/450
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/119 the label being proteinic
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• 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

Methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid, are provided. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore, energy may be transferred from the excited donor label to the acceptor label of the monomer. As a result of the energy transfer, the acceptor label emits energy, and the emitted energy is detected in order to identify the labeled monomer of the translocated polymer and to thereby sequence the polymer.

Claims

1. A method for sequencing a polymer comprising:

exciting a donor label attached to a nanopore;

translocating a polymer through the nanopore, wherein the polymer comprises a monomer labeled with an acceptor label;

transferring energy from the excited donor label to the acceptor label of the monomer after the labeled monomer passes through and exits the nanopore;

detecting the energy emitted by the acceptor label as a result of the energy transfer, wherein the energy emitted by the acceptor label is associated with a single monomer; and

deducing the polymer sequence based on the detection of the emitted energy from the acceptor label and identification of the monomer.

2. The method of claim 1, wherein the labeled polymer is threaded through the nanopore by applying an electric field through the nanopore.

3. The method of claim 1, wherein the donor label is a Quantum dot.

4. The method of claim 1, wherein the acceptor label is a fluorophore.

5. The method of claim 1, wherein a plurality of donor labels exhibiting different spectral emission maxima are attached to a nanopore and a plurality of acceptor labels having distinct absorption/emission spectra each associated with a specific monomer are attached to a polymer, and wherein each donor label and corresponding acceptor label undergo a FRET (Fö

rster Resonance Energy Transfer) interaction such that each acceptor labeled monomer of the polymer emits a specific wavelength that is detected and used to sequence the polymer.

6. The method of claim 1, wherein detecting the energy emitted by the acceptor label comprises optical detection of a specific wavelength emitted by the acceptor label and associated with a specific nucleotide.

7. The method of claim 1, wherein the polymer is a nucleic acid and the monomer is a nucleotide.

8. The method of claim 7, wherein one of four nucleotide bases is labeled and the time dependent signal arising from a nucleotide label emission is converted into a nucleic acid sequence corresponding to positions of the labeled nucleotide in the nucleic acid sequence.

9. The method of claim 8, wherein the method is repeated for each of the four nucleotides in separate samples and four partial sequences are deduced and then aligned to assemble an entire nucleic acid sequence.

10. A system for sequencing a polymer comprising:

a nanopore membrane assembly having a donor labeled nanopore which spans a lipid bilayer membrane, the nanopore membrane assembly having a negatively charged side and a positively charged side;

an aqueous buffer solution surrounding the nanopore membrane assembly;

a negative electrode positioned on the negatively charged (cis) side of the nanopore membrane assembly;

a positive electrode positioned on the positively charged (trans) side of the nanopore membrane assembly;

a polymer comprising a monomer having an acceptor label, wherein the polymer is loaded into a port positioned on the negatively charged side of the nanopore membrane assembly;

an energy source for exciting the nanopore label;

an electric field applied by the electrodes to force the labeled polymer to translocate through the nanopore from the negatively charged side to the positively charged side in a single file, wherein energy is transferred from the nanopore label to the monomer label after the labeled monomer passes through and exits the nanopore and comes in proximity to the nanopore label;

an optical detector for detecting energy emitted from the monomer label as a result of the transfer of energy from the nanopore label to the monomer label, wherein the emitted energy is associated with a single monomer and facilitates the sequencing of the polymer.

11. The system of claim 10, wherein the nanopore is labeled with one or more quantum dots and the polymer is labeled with one or more fluorophores.

12. The system of claim 10, wherein a plurality of nanopores are incorporated into the membrane in parallel, such that a plurality of polymers may be sequenced simultaneously.

13. The system of claim 10, wherein a plurality of nanopores are incorporated into a rotatable disc, such that the nanopores may be rotatably interchanged.

14. The method of claim 10, wherein the polymer is a nucleic acid and the monomer is a nucleotide.

15. A method for sequencing a polymer comprising:

exciting a donor label attached to a nanopore;

translocating a polymer through the nanopore, wherein the polymer comprises a monomer labeled with a quenching label;

quenching energy emission of the excited donor label with the quenching label of the monomer after the labeled monomer passes through and exits the nanopore;

detecting a change in photon emission by the donor label as a result of quenching the donor label with the quenching label, wherein a degree of photon emission change is associated with a single monomer of the polymer; and

deducing the polymer sequence based on the detection of the change in photon emission by the donor label and identification of the monomer.

16. The method of claim 15, wherein the polymer is a nucleic acid and the monomer is a nucleotide.