Nucleic acid sequencing by raman monitoring of molecular deconstruction

• US 6,982,165 B2
• Filed: 09/24/2001
• Issued: 01/03/2006
• Est. Priority Date: 09/24/2001
• Status: Expired due to Term

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Yamakawa, Mineo, Rao, Valluri, Berlin, Andrew, Neubauer, Gabi, Kirch, Steve
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/962,555
• Publication Number:
  US 20030058799A1
• Time in Patent Office: 1,562 Days
• Field of Search: 435/6, 435/287.2, 435/288.7, 204/182.8
• US Class Current: 435/287.2
• 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 one or more unlabeled nucleic acid molecules;

b) a flow path attached to the reaction chamber;

c) a liquid in the flow path;

d) one or more unlabeled nucleotides free in solution in the liquid at the point of nucleotide detection; and

e) a detection unit programmed to detect only the unlabeled nucleotides free in a liquid solution as they move past the detection unit, the 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 monochromoator.

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 flow path comprises a microcapillary or one or more microchannels in a chip.

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

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

8. The apparatus of claim 1, further comprising an exonuclease.

10. The apparatus of claim 1, wherein the nucleotides in solution are selected from the group consisting of adenosine monophosphate, guanosine monophosphate, cytosine monophosphate, uridine monophosphate and thymidine monophosphate.

12. The apparatus of claim 1, wherein the nucleotides in solution move past the detection unit one nucleotide at a time.

13. The apparatus of claim 1, wherein the detection unit detects unlabeled nucleotides by unenhanced Raman spectroscopy, resonance Raman scattering, coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman scattering, inverse Raman spectroscopy, stimulated gain Raman spectroscopy, hyper-Raman scattering, molecular optical laser examiner (MOLE), Raman microprobe, Raman microscopy, confocal Raman microspectrometry, three-dimensional or scanning Raman spectroscopy, Raman saturation spectioscopy, time resolved resonance Raman spectroscopy, Raman decoupling spectroscopy, or UV-Raman microscopy.

15. The apparatus of claim 1, wherein the detection unit comprises a mechanism to move the detection unit to follow the nucleotides as they move.

9. An apparatus comprising:

a) a reaction chamber to contain an immobilization surface, the immobilization surface to attach one or more unlabeled nucleic acid molecules;

b) an exonuclease in the reaction chamber;

c) at least a portion of the reaction chamber having a silver, gold, platinum, copper or aluminum mesh;

d) one or more unlabeled nucleotides free in a liquid solution at the point of nucleotide detection; and

e) a detection unit programmed to detect only the unlabeled nucleotides free in a liquid solution as they move past the detection unit, the detection unit comprising an excitation source and a Raman spectroscopic detector.

11. The apparatus of claim 9, further comprising one or more nucleic acid molecules attached to an immobilization surface.

14. The apparatus of claim 9, further comprising an exonuclease.

16. An apparatus comprising:

a) a reaction chamber to contain an immobilization surface, the immobilization surface to attach one or more unlabeled nucleic acid molecules;

b) a flow path attached to the reaction chamber, at least a portion of the flow path having a metal coating comprising silver, gold, platinum, copper or aluminum;

c) a liquid in the flow path;

d) one or more unlabeled nucleotides free in solution in the liquid at the point of nucleotide detection; and

e) a detection unit programmed to detect only the unlabeled nucleotides free in a liquid solution as they move past the detection unit, the detection unit comprising an excitation source and a Raman spectroscopic detector.