High speed parallel molecular nucleic acid sequencing

• US 20060292583A1
• Filed: 08/12/2005
• Published: 12/28/2006
• Est. Priority Date: 08/30/1999
• Status: Abandoned Application

• Current Assignee: The Government of United States of America as Represented by The Secretary of the Department of Health and Human Services
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Rubens, Denise, Schneider, Thomas D.
• Application Number:
  US11/204,367
• Publication Number:
  US 20060292583A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

A method and device is disclosed for high speed, automated sequencing of nucleic acid molecules. A nucleic acid molecule to be sequenced is exposed to a polymerase in the presence of nucleotides which are to be incorporated into a complementary nucleic acid strand. The polymerase carries a donor fluorophore, and each type of nucleotide (e.g. A, T/U, C and G) carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal (but the laser wavelength does not stimulate the acceptor fluorophore). In particular embodiments, no laser is needed if the donor fluorophore is a luminescent molecule or is stimulated by one. The emission signal from the polymerase is capable of stimulating any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added by the polymerase, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals.

Claims

1-29. -29. (canceled)

30. A substrate to which is attached a GFP-polymerase.

31. The substrate of claim 30, wherein the GFP-polymerase contains an affinity tag that attaches the GFP-polymerase to the substrate.

32. The substrate of claim 31, wherein the GFP-polymerase is attached to the substrate by a linker.

33. The substrate of claim 30, wherein the GFP-polymerase contains aequorin.

34. A method of sequencing a sample nucleic acid, comprising:

attaching a polymerase to a substrate;

allowing a sample nucleic acid and an annealed oligonucleotide to bind to the polymerase in the presence of nucleotides for incorporation into a complementary nucleic acid, wherein the polymerase and nucleotides are cooperatively labeled with donor and acceptor fluorophores that emit a unique signal when a particular nucleotide is incorporated into the complementary nucleic acid;

detecting a sequential series of the unique signals as the nucleotides are sequentially added to the complementary nucleic acid; and

converting the series of the unique signals into a nucleic acid sequence.

35-36. -36. (canceled)

37. A device for sequencing a nucleic acid molecule comprising:

a substrate to which a polymerase, oligonucleotide primer, or sample nucleic acid is attached wherein the polymerase includes a donor fluorophore;

a viewing means for viewing the polymerase;

a detection means for detecting a characteristic signal from an acceptor fluorophore carried by a corresponding nucleotide, as the nucleotide is added to the nucleic acid molecule by the polymerase;

an electromagnetic radiation source that excites the donor fluorophore but not the acceptor fluorophore; and

a decoding means for converting a series of characteristic signals into a nucleic acid sequence.

38. The device of claim 37, wherein the substrate comprises a glass microscope slide.

39. The device of claim 37, wherein the electromagnetic radiation source comprises a laser.

40. The device of claim 37, wherein the viewing means comprises a microscope objective.

41. The device of claim 37, wherein the detection means comprises a CCD camera.

42. The device of claim 37, wherein the decoding means for converting the unique signal into a nucleic acid sequence comprises a digital computer.

43. The device of claim 37, wherein the substrate comprises a three-dimensional matrix.

44. A device for sequencing a nucleic acid molecule comprising:

a glass microscope slide to which an oligonucleotide primer, sample nucleic acid, or polymerase is attached, wherein the polymerase includes a donor fluorophore;

a laser positioned to stimulate the donor fluorophore with laser light at a first wavelength range which induces the donor fluorophore to emit a signal at a second wavelength range that stimulates an acceptor flurorophore but not the donor fluorophore, and the signal emitted by the acceptor fluorophore is unique to each type of nucleotide, further wherein the first wavelength does not stimulate the acceptor fluorophore to emit the signal characteristic of the nucleotide;

a microscope objective positioned for viewing a sequence of signals emitted by the acceptor fluorophores as nucleotides are added to a sequence by the polymerase, wherein the sequence of signals corresponds to a nucleic acid sequence;

a spectrophotometer that converts the sequence of signals into a series of spectrographic signals of the acceptor fluorophore;

a CCD camera for detecting the sequence of signals; and

a digital computer which converts the sequence of signals into the nucleic acid sequence.

45. A device for sequencing a nucleic acid molecule comprising:

a glass microscope slide to which a polymerase is attached, wherein the polymerase includes a donor fluorophore;

a laser positioned to stimulate the donor fluorophore with laser light at a first wavelength range which induces the donor fluorophore to emit a signal at a second wavelength range that stimulates an acceptor fluorophore but not the donor fluorophore, and the signal emitted by the acceptor fluorophore is unique to each type of nucleotide, further wherein the first wavelength does not stimulate the acceptor fluorophore to emit the signal characteristic of the nucleotide;

a microscope objective positioned for viewing a sequence of signals emitted by the acceptor fluorophores as nucleotides are added to a sequence by the polymerase, wherein the sequence of signals corresponds to a nucleic acid sequence;

a spectrophotometer that converts the sequence of signals into a series of spectrographic signals of the acceptor fluorophore;

a CCD camera for detecting the sequence of signals; and

a digital computer which converts the sequence of signals into the nucleic acid sequence.