LABELLED NUCLEOTIDES

• US 20120202196A1
• Filed: 03/28/2012
• Published: 08/09/2012
• Est. Priority Date: 12/04/2001
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Barnes, Colin, Liu, Xiaohai, Milton, John
• Granted Patent: US 8,394,586 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.

Claims

1-25. -25. (canceled)

26. A method for controlling the incorporation of nucleotides into a polynucleotide strand, comprising the steps of:

(a) providing a nucleotide having a ribose or deoxyribose sugar moiety, wherein an enzymatically removable protecting group is attached to the 3′

oxygen atom of the ribose or deoxyribose sugar moiety of the nucleotide;

(b) providing a first enzyme to incorporate the nucleotide into the strand, whereby the protecting group prevents incorporation of another nucleotide into the strand; and

(c) providing a second enzyme to remove the protecting group to expose a 3′

-OH group on the first nucleotide, thereby allowing incorporation of another nucleotide into the strand;

(d) repeating steps (a) to (c) at least once;

thereby controlling the incorporation of nucleotides into the strand.

28. The method of claim 26 or 27, wherein the first enzyme is engineered to incorporate the protected nucleotide into the strand.

29. The method of claim 28, wherein the first enzyme is a polymerase.

30. The method of claim 29, wherein the polymerase is Klenow exo-.

31. The method of claim 28, wherein the first enzyme is a terminal transferase or a reverse transcriptase.

32. The method of claim 26 or 27, wherein the nucleotide is a ribonucleotide triphosphate.

33. The method of claim 26 or 27, wherein the nucleotide is a deoxyribonucleotide triphosphate.

34. The method of claim 26 or 27, wherein the protecting group comprises an allyl moiety.

35. The method of claim 26 or 27, wherein the protecting group comprises an azido group.

36. The method of claim 26 or 27, wherein the protecting group comprises a disulfide.

37. The method of claim 26 or 27, wherein the base of the nucleotide is a deazapurine.

38. The method of claim 26 or 27, whereby the rate of the incorporation of the nucleotides is controlled by steps (b) and (c).

39. The method of claim 26 or 27, further comprising the steps of:

(b1) detecting the incorporated nucleotide.

40. The method of claim 39, wherein the nucleotide is detected by a detectable label.

41. The method of claim 40, wherein the detectable label is a fluorophore.

42. The method of claim 41, wherein the detectable label is part of a FRET system.

43. The method of claim 39, wherein the detection is carried out with the polynucleotide on a solid support.

27. A method for controlling the incorporation of nucleotides into a polynucleotide strand, comprising the steps of:

(a) providing a nucleotide having a ribose or deoxyribose sugar moiety, wherein an enzymatically removable protecting group is attached to the 2′

oxygen atom of the ribose or deoxyribose sugar moiety of the nucleotide;

(b) providing a first enzyme to incorporate the nucleotide into the strand, whereby the protecting group prevents incorporation of another nucleotide into the strand; and

(c) providing a second enzyme to remove the protecting group to expose a 3′

-OH group on the first nucleotide, thereby allowing incorporation of another nucleotide into the strand;

(d) repeating steps (a) to (c) at least once;

thereby controlling the incorporation of nucleotides into the strand.

44. A kit, comprising:

(a) a nucleotide having a ribose or deoxyribose sugar moiety, wherein an enzymatically removable protecting group is attached to the 3′

oxygen atom of the ribose or deoxyribose sugar moiety of the nucleotide;

(b) a first enzyme that is engineered to incorporate the nucleotide into a polynucleotide strand, whereby the protecting group of the nucleotide prevents incorporation of another nucleotide into the strand; and

(c) a second enzyme to remove the protecting group to expose a 3′

-OH group on the incorporated nucleotide.

46. The kit of claim 44 or 45, wherein the protecting group comprises an allyl moiety.

47. The kit of claim 44 or 45, wherein the protecting group comprises an azido group.

48. The kit of claim 44 or 45, wherein the protecting group comprises a disulfide.

45. A kit, comprising:

(a) a nucleotide having a ribose or deoxyribose sugar moiety, wherein an enzymatically removable protecting group is attached to the 2′

oxygen atom of the ribose or deoxyribose sugar moiety of the nucleotide;

(b) a first enzyme that is engineered to incorporate the nucleotide into a polynucleotide strand, whereby the protecting group of the nucleotide prevents incorporation of another nucleotide into the strand; and

(c) a second enzyme to remove the protecting group to expose a 3′

-OH group on the incorporated nucleotide.