Labelled nucleotides

• US 9,410,200 B2
• Filed: 09/17/2015
• Issued: 08/09/2016
• 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, Liu, Xiaohai, Milton, John
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/857,671
• Publication Number:
  US 20160002719A1
• Time in Patent Office: 327 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2
• US Class Current: 1/1
• 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 of labeling a nucleic acid molecule, the method comprising:

incorporating into the nucleic acid molecule a nucleotide molecule using a polymerase, wherein the nucleotide molecule has a base that is linked to a fluorophore via a cleavable linker and the nucleotide molecule has a deoxyribose sugar moiety,

wherein the deoxyribose sugar moiety comprises a protecting group attached via the 3′

oxygen atom, and said protecting group can be modified or removed to expose a 3′

OH group, the protecting group comprising an azido group.

2. The method of claim 1, wherein the polymerase is a DNA polymerase.

3. The method of claim 2, wherein the DNA polymerase is Klenow polymerase.

4. The method of claim 1, wherein the base is a deazapurine.

5. The method of claim 1, wherein the nucleotide is a deoxyribonucleotide triphosphate.

6. The method of claim 1, wherein the cleavable linker is an acid labile linker, a photolabile linker, or a disulphide linkage.

7. The method of claim 1, wherein the protecting group is CH₂N₃.

8. The method of claim 1, further comprising removing the protecting group and cleaving the cleavable linker, said removing and cleaving step performed under identical conditions.

9. The method of claim 8, comprising detecting the fluorophore prior to the removing and cleaving step.

10. The method of claim 9, further comprising repeating the incorporating, detecting, and removing and cleaving steps at least once, thereby determining a nucleotide sequence.

11. A method of labeling a nucleic acid molecule, the method comprising:

incorporating into the nucleic acid molecule a nucleotide molecule using a polymerase, wherein the nucleotide molecule has a base that is linked to a fluorophore via a cleavable linker and the nucleotide molecule has a deoxyribose sugar moiety,

wherein the deoxyribose sugar moiety comprises a protecting group attached via the 3′

oxygen atom, and said protecting group can be modified or removed to expose a 3′

OH group, the protecting group comprising CH₂N₃.

12. The method of claim 11, wherein the polymerase is a DNA polymerase.

13. The method of claim 12, wherein the DNA polymerase is Klenow polymerase.

14. The method of claim 11, wherein the base is a deazapurine.

15. The method of claim 11, wherein the nucleotide is a deoxyribonucleotide triphosphate.

16. The method of claim 11, wherein the cleavable linker is an acid labile linker, a photolabile linker, or a disulphide linkage.

17. The method of claim 11, further comprising removing the protecting group and cleaving the cleavable linker, said removing and cleaving step performed under identical conditions.

18. The method of claim 17, comprising detecting the fluorophore prior to the removing and cleaving step.

19. The method of claim 18, further comprising the incorporating, detecting, and removing and cleaving steps at least once, thereby determining a nucleotide sequence.