Electron induced fluorescent method for nucleic acid sequencing

• US 7,157,230 B2
• Filed: 02/25/2003
• Issued: 01/02/2007
• Est. Priority Date: 08/27/2001
• Status: Expired due to Fees

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Hannah, Eric C.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US10/374,636
• Publication Number:
  US 20030165964A1
• Time in Patent Office: 1,407 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

An apparatus, compositions and related methods for sequencing a target nucleic acid are described. In certain embodiments, the apparatus is a microfluidic apparatus comprising an input chamber, microchannel, output chamber and a detection unit that is operatively connected to the microchannel. In preferred embodiments, the methods include hybridizing a target nucleic acid to one or more probe libraries, moving the hybridized target nucleic acid past the detector, and detecting bound probes. Probe libraries may comprise oligonucleotides or oligonucleotide analogs, preferably with each probe uniquely labeled. A linear order of labeled probes hybridized to the target nucleic acid can be detected and the target nucleic acid sequence deduced. In preferred embodiments, probe labels are detected by analysis of electron-induced fluorescence of probes labeled with conductive polymers.

Claims

1. A method of sequencing a target nucleic acid comprising:

obtaining the target nucleic acid;

hybridizing the nucleic acid to a probe library comprised of at least 256 distinguishably labeled different probe,

wherein the probes are labeled with conductive polymers,

wherein the conductive polymers are selected from the group consisting of polyaniline, polyphenylene-vinylene, polythiophene, polypyrrole, polyacetylene, polydiacetylene, polytriacetylene, poly-p-phenylene, polyfuran, betacarotene, a substituted polyaniline, a substituted polyphenylene-vinylene, a substituted polythiophene, a substituted polypyrrole, a substituted polyacetylene, a substituted polydiacetylene, a substituted polytriacetylene, a substituted poly-p-phenylene, a substituted polyfuran, a substituted betacarotene and a conjugated oligomer, and

wherein the substitution is an alkoxy side chain, an alkyl side chain or an aryl side chain;

separating the hybridized nucleic acid from unhybridized probes; and

detecting the order of hybridized probes along the nucleic acid.

2. The method of claim 1, further comprising moving an approximately linearly conformed hybridized nucleic acid past a detection unit.

3. The method of claim 1, further comprising exciting the probe labels with an electron beam.

4. The method of claim 3, further comprising detecting fluorescent signals emitted by the excited probe labels.

5. The method of claim 4, wherein the fluorescent signal from each different excited probe label is distinguishable.

6. The method of claim 5, further comprising determining the order of hybridized probes along the nucleic acid from the fluorescence emission spectra of the probe labels.

7. The method of claim 6, further comprising determining the sequence of the nucleic acid from the order of hybridized probes.

8. The method of claim 2, wherein the hybridized nucleic acid moves past the detection unit in a microchannel or a microcapillary.

9. The method of claim 1, wherein a single target nucleic acid molecule is sequenced at a time.

10. The method of claim 1, wherein two or more target nucleic acid molecules are sequenced at a time.

11. A method of sequencing a target nucleic acid comprising;

hybridizing the target nucleic acid to a probe library comprised of at least 256 different probes, each different probe distinguishably labeled with a conductive polymer;

separating the hybridized nucleic acid from unhybridized probes;

exciting the hybridized probes with an electron beam, wherein the excited probes of different sequence emit distinguishable fluorescent signals;

detecting an order of the hybridized probes along the nucleic acid; and

determining the target nucleic acid sequence from the order of the hybridized probes.

12. The method of claim 11, wherein the probes are 4, 5, 6, 7 or 8 nucleotides in length.

13. The method of claim 11, further comprising moving the hybridized probes and nucleic acid past a detector in an approximately linear conformation.

14. The method of claim 13, wherein the hybridized probes and nucleic acid move past the detector in a microchannel, nanochannel or microcapillary.

15. A method of sequencing a target nucleic acid comprising:

hybridizing the target nucleic acid to a probe library comprised of at least 256 different probes, each different probe distinguishably labeled with a conductive polymer;

ligating hybridized probes together;

separating the hybridized nucleic acid from unhybridized probes;

exciting the hybridized probes with an electron beam;

detecting an order of the hybridized probes along the nucleic acid; and

determining a target nucleic acid sequence from the order of the hybridized probes.

16. A method comprising:

hybridizing a target nucleic acid to a probe library comprised of at least 256 distinguishably labeled different probe;

moving the hybridized nucleic acid past a detection unit;

exciting the hybridized probes with one or more of an electron beam and an electromagnetic radiation; and

after said exciting the hybridized probes, detecting individual hybridized probes in sequential order as the individual hybridized probes of the hybridized nucleic acid move past the detection unit by detecting signals emitted by the excited hybridized probes.

17. The method of claim 16, further comprising:

determining an order of the hybridized probes along the hybridized nucleic acid from the signals emitted by the excited hybridized probes; and

determining the sequence of the nucleic acid from the order of the hybridized probes.

18. The method of claim 16, further comprising hydrodynamically focusing the hybridized nucleic acid.

19. The method of claim 16, wherein said moving the hybridized nucleic acid past the detection unit comprises flowing the hybridized nucleic acid in one selected from a microchannel and a microcapillary.

20. The method of claim 16, wherein said hybridizing comprises hybridizing the target nucleic acid to different probe that are distinguishably labeled with conductive polymers.

21. The method of claim 20, wherein said detecting the signals emitted comprises detecting light emitted by the conductive polymers after the conductive polymers have been excited with an electron beam.

22. The method of claim 16, wherein said hybridizing comprises hybridizing the target nucleic acid to different probe that are distinguishably labeled with fluorescent labels.

23. The method of claim 16, wherein said hybridizing comprises hybridizing the target nucleic acid to different probe that are each six to eight nucleotides in length.

24. The method of claim 16, wherein the excited probes emit distinguishable fluorescent signals.

25. The method of claim 16, further comprising ligating hybridized probes together.