Labelled nucleotides

• US 20100317531A1
• Filed: 07/13/2010
• Published: 12/16/2010
• Est. Priority Date: 12/04/2001
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Liu, Xiaohai, Barnes, Colin
• Granted Patent: US 8,158,346 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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

1-16. -16. (canceled)

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.

18. A method according to claim 17, further comprising the steps of

(a) providing said nucleotides;

and wherein said monitoring comprises

(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.

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 composition 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 step 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 step 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 nonincorporated 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.

26. A method according to claim 17, wherein each of the nucleotides has a base that is linked to a detectable label via a cleavable linker, wherein the cleavable linker comprises a protected hydroxyl functionality.

27. A method according to claim 17, wherein cleavage of the cleavable linker produces a hydroxyl group attached to the base.

28. A method according to claim 17, wherein cleavage of the cleavable linker produces a hydroxyl group attached to the detectable label.

29. A method according to claim 17, wherein each of the nucleotides has a ribose or deoxyribose sugar moiety.

30. A method according to claim 29, wherein the ribose or deoxyribose sugar comprises a protecting group attached via the 2′

or 3′

oxygen atom.

31. A method according to claim 17, wherein each of the nucleotides is a deoxyribonucleotide triphosphate.

32. A method according to claim 17, wherein the detectable label is a fluorophore.

33. A method according to claim 17, wherein the cleavable linker comprises a moiety cleavable using palladium

34. A method according to claim 17, wherein the cleavable linker comprises a cleavable allylic system.

35. A method according to claim 17, wherein the cleavable linker is photocleavable.

36. A method according to claim 17, wherein the detectable label and/or cleavable linker is of sufficient size to limit the incorporation of further nucleotides into a nucleic acid template strand.

37. A method according to claim 17, wherein the target single-stranded polynucleotide is immobilised on a solid support.

38. A method according to claim 37, wherein the solid support is a bead or microsphere.

39. A method according to claim 37, wherein the solid support is a glass slide.

24-25. -25. (canceled)