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

• US 7,465,578 B2
• Filed: 07/07/2004
• Issued: 12/16/2008
• 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): Strzelecka; Teresa E
• Application Number:
  US10/886,094
• Publication Number:
  US 20040248185A1
• Time in Patent Office: 1,623 Days
• Field of Search: None
• US Class Current: 435/283.1
• 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 reaction chamber to contain a polymerase and at least one nucleic acid template attached to an immobilization surface for growing a nascent strand complementary in sequence to the respective template;

a detection unit comprising an excitation source, the detection unit being adapted for identifying and quantifying nucleotide precursors in the reaction chamber to identify nucleotide precursors incorporated into the nascent strand;

a molecule dispenser for dispensing nucleotide precursors into the reaction chamber; and

an information processing and control system operably coupled to the detection unit and the molecule dispenser, for controlling nucleotide precursor release rate from the dispenser to regulate the release of nucleotide precursors by the molecule dispenser to approximately match the rate of incorporation of nucleotide precursors into the nascent strand, wherein the molecule dispenser comprises multiple molecule dispensers for dispensing four types of nucleic acid precursors, whereby the rate of release of the four types of nucleotide precursors is independently controlled.

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 data storage unit.

5. The apparatus of claim 1, wherein the reaction chamber and molecule dispenser are part of an integrated chip.

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

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