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

• US 20030059778A1
• 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 6,852,492 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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. ) An apparatus comprising:

a) a reaction chamber to contain one or more nucleic acid molecules attached to an immobilization surface; and

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

2. ) The apparatus of claim 1, wherein the reaction chamber contains a single nucleic acid molecule attached to an immobilization surface.

3. ) The apparatus of claim 1, wherein the reaction chamber contains a silver, gold, platinum, copper or aluminum mesh.

4. ) The apparatus of claim 1, further comprising a molecule dispenser.

5. ) The apparatus of claim 1, further comprising an information processing and control system.

6. ) The apparatus of claim 5, further comprising a data storage unit.

7. ) The apparatus of claim 4, wherein the reaction chamber and molecule dispenser are part of an integrated chip.

8. ) The apparatus of claim 2, further comprising:

(i) nucleotide precursors;

(ii) a synthetic reagent; and

(iii) one or more primers.

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

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

11. ) An apparatus comprising:

a) a reaction chamber, the reaction chamber containing a synthetic reagent, nucleotide precursors and a single template nucleic acid molecule attached to an immobilization surface; and

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

12. ) The apparatus of claim 11, further comprising a primer.

13. ) The apparatus of claim 11, further comprising a molecule dispenser.

14. ) The apparatus of claim 11, further comprising an information processing and control system.

15. ) The apparatus of claim 11, wherein the reaction chamber contains a silver, gold, platinum, copper or aluminum mesh.

16. ) The apparatus of claim 13, wherein the reaction chamber and molecule dispenser are part of an integrated chip.

17. ) The apparatus of claim 11, wherein the excitation source is a laser.

18. ) The apparatus of claim 11, wherein the Raman detector is a spectrometer or a monochromator.

19. ) A method of sequencing nucleic acid molecules comprising:

a) preparing a single template nucleic acid molecule;

b) inserting the template nucleic acid molecule into a reaction chamber;

c) synthesizing a complementary nucleic acid molecule from nucleotide precursors with a synthetic reagent; and

d) monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by Raman spectroscopy.

20. ) The method of claim 19, wherein a tag molecule is attached to each nucleotide precursor.

21. ) The method of claim 19, wherein the nucleotides are monitored 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.

22. ) The method of claim 19, wherein the synthetic reagent is a DNA polymerase.

23. ) The method of claim 22, further comprising adding a primer, wherein the primer is complementary in sequence to a portion of the template nucleic acid molecule.

24. ) The method of claim 23, wherein the primer is complementary to the 5′

end of the template nucleic acid molecule.

25. ) The method of claim 19, wherein the template nucleic acid molecule is attached to an immobilization surface.

26. ) The method of claim 25, wherein the template nucleic acid molecule is attached to the immobilization surface through a linker arm.

27. ) The method of claim 23, wherein the primer is attached to an immobilization surface.

28. ) A method of sequencing nucleic acid molecules comprising:

a) preparing a single template nucleic acid molecule;

b) inserting the template nucleic acid molecule into a reaction chamber;

c) synthesizing a complementary nucleic acid molecule from nucleotide precursors with a synthetic reagent; and

d) monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by fluorescence resonance energy transfer (FRET) spectroscopy.

29. ) The method of claim 28, wherein a donor tag molecule is attached to the synthetic reagent and distinguishable acceptor tag molecules are attached to each type of nucleotide precursor.

30. ) The method of claim 28, wherein one or more donor tag molecules are attached to the template nucleic acid molecule and distinguishable acceptor tag molecules are attached to each type of nucleotide precursor.