Labelled Nucleotides

• US 20030104437A1
• Filed: 08/23/2002
• Published: 06/05/2003
• Est. Priority Date: 12/04/2001
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Liu, Xiaohai, Barnes, Colin, Swerdlow, Harold
• Granted Patent: US 7,057,026 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

Abstract

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

Claims

1. A nucleotide or nucleoside molecule, having a base that is linked to a detectable label via a cleavable linker.

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

3. The molecule of claim 2, wherein the base is a deazapurine.

4. The molecule of claim 1, having a ribose or deoxyribose sugar moiety.

5. The molecule of claim 4, wherein the ribose or deoxyribose sugar comprises a protecting group attached via the 2′

or 3′

oxygen atom.

6. The molecule of claim 1, that is a deoxyribonucleotide triphosphate.

7. The molecule of claim 1, wherein the detectable label is a fluorophore.

8. The molecule of claim 1, wherein the linker is acid labile, photolabile or contains a disulphide linkage.

9. A method of labeling a nucleic acid molecule, the method comprising incorporating into the nucleic acid molecule a nucleotide or nucleoside molecule, wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker.

10. The method of claim 9, wherein said incorporating is accomplished via a terminal transferase, a polymerase or a reverse transcriptase.

11. The method of claim 9, wherein the base is a deazapurine.

12. The method of claim 9, wherein the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety, and wherein the ribose or deoxyribose sugar comprises a protecting group attached via the 2′

or 3′

oxygen atom and which can be modified or removed to expose a 3′

OH group

13. The method of claim 9, wherein the nucleotide is a deoxyribonucleotide triphosphate.

14. The method of claim 9, wherein the label is a fluorophore.

15. the method of claim 9, wherein the linker is acid labile, photolabile, or contains a disulphide linkage.

16. The method of claim 9, wherein the detectable label and/or the cleavable linker is of a size sufficient to prevent the incorporation of a second nucleotide or nucleoside into the nucleic acid molecule.

17. A method for determining the sequence of a target single-stranded polynucleotide, comprising monitoring the sequential incorporation of complementary nucleotides, wherein the nucleotides each have a base that is linked to a detectable label via a cleavable linker, and wherein the identity of each nucleotide incorporated is determined by detection of the label linked to the base, and subsequent removal of the label.

19. A method according to claim 17, wherein each of the nucleotides are brought into contact with the target sequentially, with removal of non-incorporated nucleotides prior to addition of the next nucleotide, and wherein detection and removal of the label is carried out either after addition of each nucleotide, or after addition of all four nucleotides.

20. A method according to claim 17, wherein each of the nucleotides are brought into contact with the target together simultaneously, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label.

21. A method according to claim 17, comprising a first step and a second step, wherein in the first step, a first composition comprising two of the four 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 comopsition comprising the two 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 steps and the second step are optionally repeated one or more times.

22. A method according to claim 17, comprising a first step and a second step, wherein in the first step, a composition comprising one of the four 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 the three 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 steps and the second step are optionally repeated one or more times.

23. A method according to claim 17, comprising a first step and a second step, wherein in the first step, a first composition comprising three of the four 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 composition comprising the nucleotide 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 steps and the second step are optionally repeated one or more times.

25. The kit of claim 21, further comprising an enzyme and buffers appropriate for the action of the enzyme.

18. A method for determining the sequence of a target single-stranded polynucleotide, comprising:

(a) providing nucleotides, wherein the nucleotides have a base that is linked to a detectable label via a cleavable linker, 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 target single stranded polynucleotide;

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

(d) removing the label of the nucleotide of (b); and

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

thereby determining the sequence of a target single-stranded polynucleotide.

24. A kit, comprising:

(a) individual nucleotides, wherein each nucleotide has a base that is linked to a detectable label via a cleavable linker, and wherein the detectable label linked to each nucleotide can be distinguished upon detection from the detectable label used for other three nucleotides; and

(b) packaging materials therefor.