Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication

• US 6,852,492 B2
• Filed: 09/24/2001
• Issued: 02/08/2005
• Est. Priority Date: 09/24/2001
• Status: Expired due to Fees

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Yamakawa, Mineo, Rao, Valluri, Berlin, Andrew, Neubauer, Gabi, Kirch, Steven J.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/962,288
• Publication Number:
  US 20030059778A1
• Time in Patent Office: 1,233 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/287.2, 435/287.9, 435/288.7, 356/300
• US Class Current: 435/6.13
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/60 Detection means characterised by use of a special device
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/651 Cuvettes therefore
• CPC Class Codes: G01N 2021/653 Coherent methods [CARS]
• CPC Class Codes: G01N 2021/655 Stimulated Raman
• CPC Class Codes: G01N 2021/656 Raman microprobe
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons

Abstract

The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.

Claims

1. A method of sequencing nucleic acid molecules comprising:

a) inserting multiple copies of a template nucleic acid molecule into a reaction chamber;

b) synthesizing a complementary nucleic acid molecule from nucleotide precursors with a polymerase;

c) monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule 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, wherein a tag molecule is attached to each nucleotide precursor; and

d) determining the sequence of the template nucleic acid molecule from the order of incorporation of nucleotide precursors into complementary nucleic acid molecules.

2. The method of claim 1, wherein the polymerase is a DNA polymerase.

3. The method of claim 2, further comprising adding a primer, wherein the primer is complementary in sequence to a portion of the template nucleic acid molecule.

4. The method of claim 3, wherein the primer is complementary to the 3′

end of the template nucleic acid molecule.

5. The method of claim 1, wherein the template nucleic acid molecules are attached to an immobilization surface.

6. The method of claim 5, wherein the template nucleic acid molecules are attached to the immobilization surface through a linker arm.

7. The method of claim 3, wherein the primer is attached to an immobilization surface.

8. The method of claim 1, wherein each type of nucleotide precursor is attached to a distinguishable tag molecule.

9. The method of claim 8, wherein tag molecules are selected from the group consisting of TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet cresyl blue violet, brilliant cresyl blue, para-aminobensoic acid, erythroisine, aminoacridine, cyanide, thiol, chlorine, bromine, methyl, phosphorus, sulfur and carbon nanotubes.

10. The method of claim 1, wherein tag molecules are selected from the group consisting of TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet cresyl blue violet, brilliant cresyl blue, para-aminobensoic acid, erythroisine, aminoacridine, cyanide, thiol, chlorine, bromine, methyl, phosphorus, sulfur and carbon nanotubes.

11. The method of claim 1, wherein the polymerase is a DNA polymerase, an RNA polymerase or a reverse transcriptase.

12. The method of claim 1, wherein a surface of the reaction chamber is coated with silver, gold, platinum, copper, or aluminum.

13. The method of claim 12, wherein the coated surface of the reaction chamber is opposite a detection unit.