Labelled nucleotides

• US 9,121,062 B2
• Filed: 12/02/2013
• Issued: 09/01/2015
• Est. Priority Date: 12/04/2001
• Status: Expired due to Fees

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Barnes, Colin, Liu, Xiaohai
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/094,646
• Publication Number:
  US 20140113282A1
• Time in Patent Office: 638 Days
• Field of Search: 536/23.1, 536/24.3, 536/25.3, 536/26.6, 536/25, 536/3, 435/6.1, 435/91.1
• 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 nucleoside or nucleotide comprising a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises a disulfide group and further comprises an azido group, and wherein the nucleoside or nucleotide further comprises a ribose or deoxyribose moiety which comprises a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom, and wherein the base is connected to the cleavable linker via a propargylamido or propargylamido moiety.

2. The nucleoside or nucleotide of claim 1, wherein the linker further comprises a second detectable label.

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

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

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

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

hydroxyl moiety.

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

8. The nucleoside or nucleotide of claim 6, wherein the cleavable linker prevents the incorporation of a second nucleoside or nucleotide into the ribose or deoxyribose moiety.

9. The nucleoside or nucleotide of claim 1, which is a deoxyribonucleotide triphosphate.

10. The nucleoside or nucleotide of claim 1, wherein the detectable label is a fluorophore.

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

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

13. 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 the nucleotides each independently is a nucleotide of claim 1, and wherein the identity of each nucleotide incorporated is determined by detection of the label linked to the base, and

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

wherein non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label.

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

(a) providing nucleotides, wherein the nucleotides each independently is a nucleotide of claim 1, and wherein the detectable label linked to each type of nucleotide can be distinguished upon detection from the detectable label used for other types of nucleotides;

(b) incorporating a nucleotide into the complement of the immobilized target polynucleotide and removing non-incorporated nucleotides;

(c) detecting the label of the nucleotide incorporated in (b), thereby determining the type of nucleotide incorporated;

(d) removing the label of the nucleotide detected in (c); and

(e) repeating steps (b)-(d) one or more times;

thereby determining the sequence of a target polynucleotide.

15. The method of claim 13, 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, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label, 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, 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.

16. The method of claim 13, wherein four different nucleotides are supplied simultaneously.

17. The method of claim 13, wherein the nucleotides comprise a blocking group at the 3′

position.

18. The method of claim 17, wherein the blocking group and the label are removable using a single treatment.

19. The method of claim 13, wherein the detectable label or the cleavable linker prevents the incorporation of a second nucleotide into the target polynucleotide.

20. The method of claim 13, wherein the base is a pyrimidine, purine or deazapurine.

21. The method of claim 13, wherein the nucleotides comprise a dideoxyribose moiety.

22. The method of claim 13, wherein the label is a fluorophore.

23. The method of claim 13, wherein the nucleotides are incorporated using a polymerase.

24. The method of claim 23, wherein the polymerase is an engineered polymerase.