Electron induced fluorescent method for nucleic acid sequencing

• US 20030039978A1
• Filed: 08/27/2001
• Published: 02/27/2003
• Est. Priority Date: 08/27/2001
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Hannah, Eric C.
• Granted Patent: US 6,767,731 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 composition comprising a probe library, the library consisting of at least 256 different probes, wherein each different probe is distinguishably labeled.

2. The composition of claim 1, wherein the probes are oligonucleotides, chemically modified oligonucleotides, oligonucleotide analogs or peptide nucleic acids.

3. The composition of claim 1, wherein the probes are labeled with conductive polymers.

4. The composition of claim 1, wherein the library consists of about 500, about 1000, about 2000, about 4000, about 8000, about 16,000, about 32,000 or about 64,000 different probes.

5. The composition of claim 1, wherein the probes comprise random sequences.

6. The composition of claim 5, wherein the random sequences are 4, 5, 6, 7 or 8 bases long.

7. The composition of claim 5, wherein the random sequences are attached to one or more constant sequences.

8. The composition of claim 7, wherein the constant sequences are 1, 2, 3 or 4 bases long.

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

a) obtaining a target nucleic acid;

b) hybridizing the nucleic acid to a probe library, the library consisting of at least 256 different probes, wherein each different probe is distinguishably labeled;

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

d) detecting the order of hybridized probes along the nucleic acid.

10. The method of claim 9, further comprising moving the hybridized nucleic acid past a detection unit, wherein the nucleic acid is in an approximately linear conformation.

11. The method of claim 9, wherein the probes are labeled with conductive polymers.

12. The method of claim 11, wherein the probe labels are excited with an electron beam.

13. The method of claim 12, wherein the excited probe labels emit a fluorescent signal.

14. The method of claim 13, wherein the fluorescence emission spectra of the probe labels are used to detect the order of hybridized probes along the nucleic acid.

15. The method of claim 10, wherein the hybridized nucleic acid moves past the detection unit in a microchannel or a microcapillary.

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

17. The method of claim 9, wherein two or more target nucleic acid molecules are sequenced at a time.

18. 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 an electron beam source and a detector.

19. The apparatus of claim 18, further comprising an information processing and control system operably coupled to the detector.

20. The apparatus of claim 18, further comprising a fluidic focusing region between the input chamber and the microchannel.

21. The apparatus of claim 18, further comprising a target nucleic acid hybridized to multiple labeled probes, each probe distinguishably labeled.

22. The apparatus of claim 21, wherein the probes are labeled with conductive polymer molecules.

23. The apparatus of claim 20, wherein the fluidic focusing region contains a separating comb.

24. The apparatus of claim 18, wherein the electron beam source is a micro column electron beam source.

25. The apparatus of claim 24, wherein the micro column electron beam source is as shown in FIG. 5.

26. The apparatus of claim 18, further comprising an array of microfluidic sequencing units.