Methods to increase nucleotide signals by Raman scattering

• US 7,238,477 B2
• Filed: 09/12/2003
• Issued: 07/03/2007
• Est. Priority Date: 09/24/2001
• Status: Expired due to Fees

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Yamakawa, Mineo, Su, Xing, Rao, Valluri, Berlin, Andrew A., Chan, Selena, Neubauer, Gabi, Kirch, Steven J., Sundararajan, Narayanan, Koo, Tac-Woong
• Primary Examiner(s): Fredman; Jeffrey
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US10/660,902
• Publication Number:
  US 20040126790A1
• Time in Patent Office: 1,390 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer

Abstract

The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.

Claims

1. A method comprising:

a) obtaining nucleotides covalently linked to gold or silver, or gold or silver nanoparticle(s), wherein the nucleotide and nanoparticles are linked via a terminal reactive cross-linking group, selected from the group consisting of epoxide groups, azido groups, triazine groups, arylazido groups and diazo groups;

b) synthesizing one or more nucleic acid molecules comprising the gold or silver, or gold or silver nanoparticles;

c) immobilizing the nucleic acid molecule of step (b) on a solid substrate;

d) sequentially releasing nucleotides from one end of one or more nucleic acid molecules via an exonuclease;

e) identifying the released unlabeled nucleotides in a buffer comprising an alkali-metal halide salt by Raman spectroscopy; and

f) determining the sequence of the nucleic acid molecule.

2. The method of claim 1, wherein single molecules of nucleotides are identified by Raman spectroscopy.

3. The method of claim 2, wherein a single nucleic acid molecule is sequenced.

4. The method of claim 1, wherein multiple nucleic acid molecules of the same sequence or multiple nucleic acid molecules of different sequences are sequenced simultaneously.

5. The method of claim 1, wherein the alkali-metal halide salt is selected from the group consisting of MgCl, CaCl. NaF, KBr, LiI, and LiCl.

6. The method of claim 5, wherein the alkali-metal halide salt is LiCl.

7. The method of claim 1, wherein the linker compound is 3-glycidoxypropyltrimethoxysilane(GOP).

8. The method of claim 1, wherein the released nucleotides are identified by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS).

9. The method of claim 1, further comprising separating the nucleotides from the one or more nucleic acid molecules by transferring the released nucleotides through a microfluidic channel.

10. The method of claim 9, wherein microfluidic channel is a metal coated channel.

11. The method of claim 10, wherein the metal is silver, gold, platinum, copper, or aluminum.

12. The method of claim 11, wherein the nanoparticle and microfluidic channel comprise silver.