SHORT CYCLE METHODS FOR SEQUENCING POLYNUCLEOTIDES

• US 20090191565A1
• Filed: 02/13/2009
• Published: 07/30/2009
• Est. Priority Date: 11/12/2003
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Phillip Richard, Harris, Timothy, Lapidus, Stanley
• Granted Patent: US 7,897,345 B2
• Time in Patent Office: Days
• US Class Current: 435/6.16
• 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 2527/113 Time

Abstract

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or fill completion.

Claims

1-26. -26. (canceled)

27. A method for simultaneously sequencing plural nucleic acid templates, the method comprising:

a) exposing simultaneously plural single molecule nucleic acid templates immobilized on a surface, each of which is hybridized to a primer, to (i) a polymerase capable of catalyzing nucleotide addition to said primers hybridized to said templates, and (ii) one or more optically detectable labeled nucleotides that are not chain terminating nucleotides, under reaction conditions and for a period of time such that, on average, only one of fewer nucleotides is added to each said primer by said polymerase to produce plural extended primers incorporating said nucleotides; and

b) optically identifying

     1) which of the individual templates incorporates a nucleotide in step (a) and

     2) the type of the nucleotide added.

28. The method of claim 27, wherein the templates are individually optically resolvable.

29. The method of claim 27, wherein the reaction conditions are statistically insufficient for incorporation of more nucleotides than are resolvable by a detection system used in step b).

30. The method of claim 27, wherein the reaction in a) is controlled by modifying the polymerase activity.

31. The method of claim 27, wherein the reaction in a) is controlled by the addition of cofactors.

32. The method of claim 27, wherein the reaction in a) is controlled by the label on the nucleotide(s).

33. The method of claim 27, wherein the polymerase is a modified polymerase.

34. The method of claim 27, wherein the reaction in a) is controlled by the temperature.

35. The method of claim 27, wherein the optical identification comprises detection of fluorescent emission.

36. The method of claim 27, wherein the fluorescence resonance energy transfer (FRET) is employed for detection.

37. The method of claim 36, wherein the FRET donor attaches to the polymerase.

38. The method of claim 27, wherein a DNA-binding protein is used to allow FRET.

39. The method of claim 27, wherein the optical label is selected from cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.

40. The method of claim 27, wherein the optically labeled nucleotide(s) are a natural or non-natural nucleotide chosen from a nucleotide analogue and a modified nucleotide.

41. The method of claim 27, wherein the optically labeled nucloetide(s) comprises a deoxynucleotide selected from dATP, dTTP, dUTP, dCTP, and dGTP.

42. The method of claim 27, wherein the optical label is a fluorescent label which is linked by a cleavable linker to the deoxyribose of the nucleotide(s).

43. A method for simultaneously sequencing individually optically resolvable plural nucleic acid templates, the method comprising:

a) exposing simultaneously plural single molecule nucleic acid templates immobilized on a surface, each of which is hybridized to a primer, to (i) a polymerase capable of catalyzing nucleotide addition to said primers hybridized to said templates, and (ii) one or more optically detectable labeled nucleotides that are not chain terminating nucleotides thereby the nucleotides are added to each of said primers by said polymerase to produce plural extended primers incorporating said nucleotides; and

b) optically identifying

     1) which of the individual templates incorporated a nucleotide in step (a) and

     2) the type of the nucleotide added, wherein the reaction conditions in a) are insufficient for incorporation of more nucleotides than are resolvable by a detection system used in step b).