Labelled nucleotides

• US 10,519,496 B2
• Filed: 03/27/2017
• Issued: 12/31/2019
• Est. Priority Date: 12/04/2001
• Status: Expired due to Fees

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Barnes, Colin Lloyd, Liu, Xiaohai, Wu, Xiaolin, Milton, John
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/470,801
• Publication Number:
  US 20170204458A1
• Time in Patent Office: 1,009 Days
• Field of Search: 435 61, 435 911, 536 41, 536 231, 536 243, 536 253, 536 266
• 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 nucleotide comprising a base linked to a detectable label via a cleavable linker comprising an azido group, wherein the nucleotide further comprises a ribose or deoxyribose moiety, said ribose or deoxyribose comprising an azido protecting group attached to the 2′

or 3′

oxygen atom.

2. The nucleotide of claim 1, wherein the base is a purine, a deazapurine, or a pyrimidine.

3. The nucleotide of claim 2, wherein the base is attached to the cleavable linker at the 7-position of the purine or the 5-position of the pyrimidine.

4. The nucleotide of claim 1, wherein same chemical conditions may be used to effect cleavage of the cleavable linker and to remove the azido protecting group.

5. The nucleotide of claim 1, wherein the ribose or deoxyribose moiety comprises an unprotected 3′

hydroxyl moiety.

6. The nucleotide of claim 5, wherein the detectable label prevents the incorporation of a second nucleotide into the ribose or deoxyribose moiety.

7. The nucleotide of claim 5, wherein the cleavable linker prevents the incorporation of a second nucleotide into the ribose or deoxyribose moiety.

8. The nucleotide of claim 1, which is a deoxyribonucleotide triphosphate and the azido protecting group is attached to the 3′

oxygen atom.

9. The nucleotide of claim 8, wherein the detectable label is a fluorophore.

10. An oligonucleotide comprising at least one nucleotide of claim 1.

11. The oligonucleotide of claim 10, wherein at least one nucleotide of claim 1 is present at a terminal position of the oligonucleotide.

12. A method for determining the sequence of an immobilized target polynucleotide, comprising:

(a) monitoring the sequential incorporation of nucleotides complementary to the immobilized target polynucleotide, wherein each of the nucleotides independently is a nucleotide of claim 1, and wherein the identity of each nucleotide incorporated is determined by detection of the detectable label linked to the base; and

(b) removing the detectable label from the base by cleavage of the cleavable linker;

wherein non-incorporated nucleotides are removed prior to detection and the detectable label is removed subsequent to detection.

13. The method of claim 12, comprising a first step and a second step, wherein in the first step, a first composition comprising two different nucleotides is brought into contact with the target polynucleotide, non-incorporated nucleotides are removed prior to detection and the detectable label is removed subsequent to detection, and wherein in the second step, a second composition comprising two different nucleotides not included in the first composition is brought into contact with the target polynucleotide, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label, and wherein the first step and the second step are optionally repeated one or more times.

14. The method of claim 12, wherein four different nucleotides are supplied simultaneously.

15. The method of claim 12, wherein each of the nucleotides comprises the azido protecting group at the 3′

oxygen atom of the ribose or deoxyribose.

16. The method of claim 15, wherein the azido protecting group and the detectable label are removable using a single treatment.

17. The method of claim 12, wherein the detectable label or the cleavable linker prevents the incorporation of a second nucleotide into the target polynucleotide.

18. The method of claim 12, wherein the base is a pyrimidine, purine or deazapurine.

19. The method of claim 12, wherein each of the nucleotides comprises a deoxyribose moiety.

20. The method of claim 12, wherein the detectable label is a fluorophore.