Electron induced fluorescent method for nucleic acid sequencing

• US 6,767,731 B2
• Filed: 08/27/2001
• Issued: 07/27/2004
• Est. Priority Date: 08/27/2001
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Hannah, Eric C.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/940,228
• Publication Number:
  US 20030039978A1
• Time in Patent Office: 1,065 Days
• Field of Search: 422/82.05, 422/82.08, 435/287.2, 435/288.5, 435/288.6, 435/288.7, 204/403.13, 204/603, 313/306, 313/309
• US Class Current: 435/287.2
• 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 nucleic acid sequencing apparatus comprising:

a) an input chamber;

b) a microchannel in fluid communication with the input chamber; and

c) a detection unit operably coupled to the microchannel, the detection unit comprising a detector and a micro column electron beam source comprising;

an electron source positioned at the opening of an evacuated column;

a low-voltage gate electrode at the opening of the evacuated column;

aligned high-voltage acceleration, deflection and focusing electrodes perpendicular to the long axis of the evacuated column; and

an electron transparent membrane to seal the far end of the evacuated column.

2. The apparatus of claim 1, further comprising an information processing and control system operably coupled to the detector.

3. The apparatus of claim 1, further comprising a fluidic focusing region between the input chamber and the microchannel.

4. The apparatus of claim 1, further comprising a target nucleic acid hybridized to multiple labeled probes, each probe distinguishably labeled.

5. The apparatus of claim 4, wherein the probes are labeled with conductive polymer molecules.

6. The apparatus of claim 3, wherein the fluidic focusing region contains a separating comb.

7. The apparatus of claim 1, further comprising an array of microfluidic sequencing units.

8. The apparatus of claim 5, wherein the conductive polymer molecules 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.

9. The apparatus of claim 8, wherein the substitution is an alkoxy side chain, an alkyl side chain or an aryl side chain.

10. The apparatus of claim 1, wherein said electron transparent membrane is positioned so that an electron beam can be focused onto the microchannel coupled to the membrane.

11. The apparatus of claim 10, wherein said detection unit comprises an array of optical detectors.

12. The apparatus of claim 7, wherein each microfluidic sequencing unit comprises:

a micro column electron beam source;

inlet and outlet ports;

input and output chambers;

a fluidic focusing region;

a microchannel; and

inlet and outlet exit ports.

13. The apparatus of claim 1, wherein said detector comprises a spectrophotometer, photomultiplier tube, charge-coupled device, avalanche photodiode or double grating monochromator.