SYSTEM AND METHODS FOR NUCLEIC ACID SEQUENCING OF SINGLE MOLECULES BY POLYMERASE SYNTHESIS

• US 20090082212A1
• Filed: 10/24/2007
• Published: 03/26/2009
• Est. Priority Date: 12/14/1998
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience Labs (L'Oreal SA)
• Inventors: Williams, John G.K.
• Granted Patent: US 8,192,961 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Claims

1-30. -30. (canceled)

31. A method for identifying nucleotides in a target nucleic acid sequence, said method comprising:

a) providing a complex of a polymerase, a template nucleic acid and a primer sequence immobilized on a solid support;

b) contacting said complex with one or more labeled nucleotides, wherein the one of more labeled nucleotides are unblocked at the 3′

OH; and

c) detecting incorporation of the one or more nucleotides in a primer extension reaction.

32. The method of claim 31, wherein the contacting step comprises contacting the complex with more than one nucleotide at a time.

33. The method of claim 32, wherein the one or more nucleotides comprise nucleotides labeled on the γ

-phosphate.

34. The method of claim 33, wherein said γ

-phosphate label is cleaved upon incorporation.

35. The method of claim 34, wherein the cleaved label is washed away.

36. The method of claim 31, wherein said label is a fluorescent label.

37. The method of claim 31, further comprising washing unincorporated labeled nucleotides away from said complex.

38. The method of claim 31, wherein said complex is immobilized as an array of individual, single molecules.

39. The method of claim 31, wherein the surface of the solid support comprises an array of locations each bearing a complex.

40. The method of claim 39, wherein each member of said array is located in a defined location.

41. The method of claim 39, wherein each member of said array is different.

42. The method of claim 39, wherein said array of molecules is addressable.

43. The method of claim 31, wherein said target nucleic acid is a single nucleotide polymorphism.

44. The method of claim 31, wherein detecting a unique signal from the labeled nucleotide involves using a system or device selected from the group that consists of a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge couple device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.

45. The method of claim 31, wherein the detection is a sequential detection of different types of nucleotides to provide the sequence of said target nucleic acid.

46. The method of claim 31, wherein a solution contacting said solid support flows past the immobilized polymerase or target nucleic acid.

47. The method of claim 31, wherein said detecting comprises acquiring a sequence of images.

48. The method of claim 31, wherein said sequence of images is analyzed to track single molecules.

49. The method of claim 31, wherein said detecting is carried out by a mechanism that provides low background and enables the detection of single molecules.

50. The method of claim 31, wherein said detecting is carried out by an evanescent optical field.

51. The method of claim 31, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.