Electron induced fluorescent method for nucleic acid sequencing

• US 20030165964A1
• Filed: 02/25/2003
• Published: 09/04/2003
• Est. Priority Date: 08/27/2001
• Status: Active Grant

• Current Assignee: Eric C. Hannah
• Original Assignee: Eric C. Hannah
• Inventors: Hannah, Eric C.
• Granted Patent: US 7,157,230 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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:

a) obtaining a target nucleic acid;

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

c) separating the hybridized nucleic acid from unhybridized probes; and

d) 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, wherein the probes are labeled with conductive polymers.

4. The method of claim 3, 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.

5. The method of claim 4, wherein the substitution is an alkoxy side chain, an alkyl side chain or an aryl side chain.

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

7. The method of claim 6, further comprising detecting fluorescent signals emitted by the excited probe labels.

8. The method of claim 7, wherein the fluorescent signal from each different excited probe label is distinguishable.

9. The method of claim 8, further comprising determining the order of hybridized probes along the nucleic acid from the fluorescence emission spectra of the probe labels.

10. The method of claim 9, further comprising determining the sequence of the nucleic acid from the order of hybridized probes.

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

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

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

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

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

b) separating the hybridized nucleic acid from unhybridized probes;

c) exciting the hybridized probes with an electron beam; and

d) detecting the order of hybridized probes along the nucleic acid; and

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

15. The method of claim 14, wherein the probes are 4, 5, 6, 7 or 8 nucleotides in length.

16. The method of claim 14, wherein the excited probes of different sequence emit distinguishable fluorescent signals.

17. The method of claim 14, further comprising moving the hybridized probes and nucleic acid past a detector in an approximately linear conformation.

18. The method of claim 17, wherein the hybridized probes and nucleic acid move past the detector in a microchannel, nanochannel or microcapillary.

19. The method of claim 14, further comprising ligating hybridized probes together.