Labelled nucleotides

• US 8,394,586 B2
• Filed: 03/28/2012
• Issued: 03/12/2013
• Est. Priority Date: 12/04/2001
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Barnes, Colin, Liu, Xiaohai, Milton, John
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/432,989
• Publication Number:
  US 20120202196A1
• Time in Patent Office: 349 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 422/430
• 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. 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;

wherein the protecting group comprises an azido group or a disulfide.

3. The method of claim 1 or 2, wherein the first enzyme is engineered to incorporate the protected nucleotide into the strand.

4. The method of claim 3, wherein the first enzyme is a polymerase.

5. The method of claim 4, wherein the polymerase is Klenow exo-.

6. The method of claim 3, wherein the first enzyme is a terminal transferase or a reverse transcriptase.

7. The method of claim 1 or 2, wherein the nucleotide is a ribonucleotide triphosphate.

8. The method of claim 1 or 2, wherein the nucleotide is a deoxyribonucleotide triphosphate.

9. The method of claim 1 or 2, wherein the protecting group comprises an azido group.

10. The method of claim 1 or 2, wherein the protecting group comprises a disulfide.

11. The method of claim 1 or 2, wherein the base of the nucleotide is a deazapurine.

12. The method of claim 1 or 2, whereby the rate of the incorporation of the nucleotides is controlled by steps (b) and (c).

13. The method of claim 1 or 2, further comprising the steps of:

(b

     1) detecting the incorporated nucleotide.

14. The method of claim 13, wherein the nucleotide is detected by a detectable label.

15. The method of claim 14, wherein the detectable label is a fluorophore.

16. The method of claim 15, wherein the detectable label is part of a FRET system.

17. The method of claim 13, wherein the detection is carried out with the polynucleotide on a solid support.

2. 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;

wherein the protecting group comprises an azido group or a disulfide.

18. 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;

wherein the protecting group comprises an azido group or a disulfide.

20. The kit of claim 18 or 19, wherein the protecting group comprises an azido group.

21. The kit of claim 18 or 19, wherein the protecting group comprises a disulfide.

19. 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;

wherein the protecting group comprises an azido group or a disulfide.