Nucleic acid sequencing by raman monitoring of molecular deconstruction

• US 20030058799A1
• Filed: 09/24/2001
• Published: 03/27/2003
• Est. Priority Date: 09/24/2001
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Yamakawa, Mineo, Rao, Valluri, Berlin, Andrew, Neubauer, Gabi, Kirch, Steven J.
• Granted Patent: US 6,982,165 B2
• Time in Patent Office: Days
• US Class Current: 370/241
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer

Abstract

The disclosed methods, apparatus and compositions are of use for nucleic acid sequencing. More particularly, the methods and apparatus concern sequencing single molecules of single stranded DNA or RNA by exposing the molecule to exonuclease activity, removing free nucleotides one at a time from one end of the nucleic acid, and identifying the released nucleotides by Raman spectroscopy or FRET.

Claims

1. ) An apparatus comprising:

a) a reaction chamber to contain an immobilization surface, the immobilization surface to attach a single nucleic acid molecule;

b) a flow path attached to the reaction chamber; and

c) a detection unit comprising an excitation source and a Raman spectroscopic detector.

2. ) The apparatus of claim 1, wherein the excitation source is a laser.

3. ) The apparatus of claim 1, wherein the Raman detector is a spectrometer or monochromator.

4. ) The apparatus of claim 1, further comprising (i) an information processing system;

and (ii) a database.

5. ) The apparatus of claim 1, wherein the immobilization surface is a magnetic bead, a non-magnetic bead, a conical surface, a planar surface or a curved surface.

6. ) The apparatus of claim 1, wherein the flow path comprises a microcapillary or one or more microchannels in a chip.

7. ) The apparatus of claim 1, wherein a portion of the flow path is coated with silver, gold, platinum, copper or aluminum.

8. ) The apparatus of claim 1, wherein the flow path contains a silver, gold, platinum, copper or aluminum mesh.

9. ) The apparatus of claim 1, further comprising a deconstruction reagent.

10. ) An apparatus comprising:

a) a reaction chamber to contain an immobilization surface, the immobilization surface to attach a single nucleic acid molecule; and

b) a detection unit comprising an excitation source and a Raman spectroscopic detector.

11. ) The apparatus of claim 10, further comprising (i) an information processing system;

and (ii) a database.

12. ) The apparatus of claim 10, wherein the excitation source is a laser.

13. ) The apparatus of claim 10, wherein the Raman detector is a spectrometer or monochromator.

14. ) The apparatus of claim 10, wherein the immobilization surface is a magnetic bead, a non-magnetic bead, a conical surface, a planar surface or a curved surface.

15. ) The apparatus of claim 10, further comprising a silver, gold, platinum, copper or aluminum mesh in the reaction chamber.

16. ) The apparatus of claim 10, further comprising a deconstruction reagent.

17. ) A method of sequencing nucleic acids comprising:

a) preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;

b) sequentially removing nucleotides from the unattached end of the nucleic acid molecule;

c) separating the nucleotides from the nucleic acid molecule; and

d) detecting the nucleotides by Raman spectroscopy.

18. ) The method of claim 17, wherein the nucleotides are removed from the unattached end of the nucleic acid molecule by exonuclease activity.

19. ) The method of claim 17, wherein a tag molecule is attached to each nucleotide after the nucleotide is removed from the nucleic acid molecule.

20. ) The method of claim 17, wherein the nucleotides are detected by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering.

21. ) A method of sequencing nucleic acids comprising:

a) preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;

b) sequentially removing nucleotides from the unattached end of the nucleic acid molecule; and

c) quantifying the nucleotides in the reaction chamber by Raman spectroscopy.

22. ) The method of claim 21, wherein the nucleotides are removed from the unattached end of the nucleic acid molecule by exonuclease activity.

23. ) The method of claim 21, wherein a tag molecule is attached to each nucleotide after the nucleotide is removed from the nucleic acid molecule.

24. ) The method of claim 21, wherein the nucleotides are detected by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering.

25. ) A method of sequencing nucleic acids comprising:

a) preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;

b) sequentially removing nucleotides from the unattached end of the nucleic acid molecule; and

c) detecting the nucleotides by fluorescence resonance energy transfer (FRET) spectroscopy.

26. ) The method of claim 25, wherein the nucleotides are attached to acceptor molecules after they are removed from the nucleic acid molecule.

27. ) The method of claim 26, wherein the nucleotides are removed by an exonuclease.

28. ) The method of claim 27, wherein one or more donor molecules is attached to the exonuclease.