Labelled nucleotides

• US 7,785,796 B2
• Filed: 07/23/2009
• Issued: 08/31/2010
• Est. Priority Date: 12/04/2001
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Milton, John, Liu, Xiaohai, Barnes, Colin
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/460,741
• Publication Number:
  US 20100028885A1
• Time in Patent Office: 404 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/26.6
• US Class Current: 435/6.11
• 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 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 have a base that is linked to a detectable label via a cleavable linker, wherein the cleavable linker comprises an allylic system 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.

3. The method according to claim 1, wherein non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label.

4. The method according to claim 1, 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.

5. The method according to claim 1, comprising a first step and a second step, wherein in the first step, a composition comprising one type of 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 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.

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

7. The method according to claim 1, wherein four different nucleotides are supplied simultaneously.

8. The method according to claim 1, wherein the allylic system is cleaved using a catalyst comprising palladium.

9. The method according to claim 1, wherein the nucleotides comprise a blocking group at the 3′

position.

10. The method according to claim 9, wherein the blocking group and the label are removable using a single treatment.

11. The method according to claim 1, wherein the nucleotides comprise a dideoxyribose moiety.

12. The method according to claim 1, wherein the base is a pyrimidine, purine or deazapurine.

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

14. The method according to claim 1, wherein the nucleotides are incorporated using a polymerase.

15. The method according to claim 14, wherein the polymerase is an engineered polymerase.

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

(a) providing nucleotides, wherein the nucleotides have a base that is linked to a detectable label via a cleavable linker, wherein the cleavable linker comprises an allylic system, 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;

(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.