Nucleic acid sequencing using an optically labeled pore

• US 6,528,258 B1
• Filed: 09/01/2000
• Issued: 03/04/2003
• Est. Priority Date: 09/03/1999
• Status: Expired due to Term

• Current Assignee: Lifebeam Technologies, Inc.
• Original Assignee: Lifebeam Technologies, Inc.
• Inventors: Russell, Terence S.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/653,046
• Time in Patent Office: 914 Days
• Field of Search: 435/6, 536/24.1, 536/23.1
• US Class Current: 435/6.15
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• 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 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48707 by electrical means G01N33/49, G01N33/493 take precedence

Abstract

The present invention provides a system for analyzing polymer molecules by detecting their effects on an optical agent. Certain preferred embodiments of the invention involve the analysis of polynucleotide molecules through detection of their quenching effects on a fluorescent reporter.

Claims

1. An apparatus comprising:

an alpha-hemolysin pore;

an optical agent associated with the pore, the optical agent being selected and arranged so that, when a nucleic acid polymer is passed through the pore, each base of the polymer interacts with the optical agent in a characteristic way, resulting in a characteristic change in an optical property of the optical agent; and

a detector selected and arranged to detect the characteristic changes so that, as the nucleic acid polymer passes through the pore, the bases are individually identified by the detected characteristic changes.

5. The apparatus of claim 1, 2, 3, or 4, wherein the optical agent comprises a fluorophore.

6. The apparatus of claim 5, wherein the characteristic change comprises quenching of fluorescence.

7. The apparatus of claim 6, wherein the quenching results from electron transfer and the pore and optical agent are arranged with respect to one another so that, when a polymer passes through the pore, its monomers pass within 10-100 Angstroms of the optical agent.

8. The apparatus of claim 1, 2, 3, or 4 wherein the optical agent has sufficient stability to allow analysis of polymers at least 1000 monomer units long.

9. The apparatus of claim 1, 2, 3, or 4, wherein the pore comprises at least one protein.

10. The apparatus of claim 1, 2, 3, or 4 wherein the optical agent comprises a quantum dot.

11. The apparatus of claim 1, 2, 3, or 4, wherein the optical agent is linked to the pore through covalent or non-covalent interactions.

2. An apparatus comprising:

a biological pore defining a channel dimensioned to accept a nucleic acid polymer so that a single nucleic acid strand passes in linear fashion through the pore;

an optical agent associated with the pore, the optical agent being selected and arranged to that, when a nucleic acid polymer is passed through the pore, each base of the polymer interacts with the optical agent in a characteristic way, resulting in a characteristic change in an optical property of the optical agent; and

a detector selected and arranged to detect the characteristic changes so that, as the nucleic acid polymer passes through the pore, the bases are individually identified by the detected characteristic changes.

3. An apparatus comprising:

a biological pore defining a channel dimensioned to accept a nucleic acid polymer so that a single nucleic acid strand passes in linear fashion through the pore;

an optical agent associated with the pore, the optical agent being selected and arranged to that, when an unlabeled nucleic acid polymer is passed through the pore, each nucleotide residue interacts with the optical agent in a characteristic way, resulting in a characteristic change in an optical property of the optical agent; and

a detector selected and arranged to detect the characteristic changes so that, as the nucleic acid polymer passes through the pore, the -nucleotide residues are individually identified by the detected characteristic changes.

4. An apparatus comprising:

a pore defining a channel dimensioned to accept a nucleic acid polymer so that the nucleic acid polymer passes in linear fashion through the pore;

an optical agent associated with the pore, the optical agent being selected and arranged to interact with individual monomers of the nucleic acid polymer in a characteristic way, resulting in a characteristic change in an optical property of the optical agent; and

a detector selected and arranged to detect the characteristic changes so that, as the nucleic acid polymer passes through the pore the monomers are individually identified in their linear order by the detected characteristic changes.