Labelled nucleotides

Patent metadata

Field	Value
Publication number	US-9605310-B2
Application number	US-201514821566-A
Country	US
Kind code	B2
Filing date	Aug 7, 2015
Priority date	Dec 4, 2001
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

Abstract

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

First claim

The invention claimed is:

1. 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 vial a cleavable linker comprising an azido group, wherein each of the nucleotides has a ribose or deoxyribose sugar moiety and the ribose or deoxyribose sugar moiety comprises an azido protecting group attached via the 2′ or 3′ oxygen atom, 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 and the protecting group under a single set of conditions.

2. The method according to claim 1 , 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.

3. The method according to claim 1 ,
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.

4. The method according to claim 1 , 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.

5. The method according to claim 1 , 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.

6. The method according to claim 1 , comprising a first step and a second step,
wherein in the first step, a first 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.

7. The method according to claim 1 , 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 second 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 step and the second step are optionally repeated one or more times.

8. The method according to claim 1 , 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.

9. The method according to claim 1 , wherein cleavage of the cleavable linker produces a hydroxyl group attached to the base.

10. The method according to claim 1 , wherein cleavage of the cleavable linker produces a hydroxyl group attached to the detectable label.

11. The method according to claim 1 , wherein each of the nucleotides is a deoxyribonucleotide triphosphate.

12. The method according to claim 1 , wherein the detectable label is a fluorophore.

13. The method according to claim 1 , 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.

14. The method according to claim 1 , wherein the target single-stranded polynucleotide is immobilised on a solid support.

15. The method according to claim 14 , wherein the solid support is a bead or microsphere.

16. The method according to claim 14 , wherein the solid support is a glass slide.

Assignees

• Illumina Cambridge Ltd

Inventors

• Balasubramanian Shankar
• Barnes Colin
• Liu Xiaohai

Classifications

• C12Q1/6823

  Release of bound markers · CPC title

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title