Labelled nucleotides

• US 8,148,064 B2
• Filed: 07/19/2010
• Issued: 04/03/2012
• Est. Priority Date: 12/04/2001
• Status: Expired due to Fees

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Barnes, Colin, Liu, Xiaohai, Milton, John
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/804,296
• Publication Number:
  US 20110183327A1
• Time in Patent Office: 624 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 536/26.6
• US Class Current: 435/6
• 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 method for detecting the extension of a primer annealed to a target polynucleotide, comprising:

(a) providing at least one nucleoside comprising

(1) a label attached via a first linker to the nucleoside base, and

(2) a protecting group attached via a second linker to the 2′

or 3′

oxygen of the nucleoside sugar,

wherein the first and second linkers are both cleavable under a single set of conditions;

(b) extending the annealed primer with the nucleoside only if the nucleoside base is complementary to the base at the corresponding position of the target polynucleotide;

(c) detecting the presence of the label on the extended primer; and

(d) incubating the extended primer under the single set of cleavage conditions, whereby both the first and second linkers are cleaved;

thereby detecting the extension of the primer.

2. The method of claim 1, wherein the target polynucleotide is immobilized.

3. The method of claim 1, wherein step (a) provides at least two different nucleosides, each having a different label, and the different labels are distinguishable in step (c).

4. The method of claim 3, wherein step (a) provides four different nucleosides.

5. The method of claim 1, wherein the nucleoside provided in step (a) is phosphorylated as a nucleotide.

6. The method of claim 5, wherein the nucleotide provided in step (a) comprises three phosphate groups.

7. The method of claim 1, wherein the first and/or second linkers comprise an azide.

8. The method of claim 1, wherein the first and/or second linkers comprise an allylic group.

9. The method of claim 1, wherein extending step (b) is performed by a DNA polymerase.

10. The method of claim 1, wherein extending step (b) is performed by a reverse transcriptase.

11. The method of claim 1, wherein extending step (b) is performed by a terminal transferase.

12. The method of claim 1, wherein after step (b), any non-incorporated nucleosides are separated from the extended primers.

13. The method of claim 1, wherein step (d) removes the protecting group to expose a hydroxyl.

14. The method of claim 1, further comprising:

(e) repeating steps (a), (b), (c), and (d) sequentially.

15. The method of claim 14, wherein step (e) is repeated more than once.

16. The method of claim 14, wherein steps (a)-(d) are initially performed with one nucleoside and steps (a)-(d) in step (e) are performed with a different nucleoside.

17. The method of claim 16, wherein two nucleosides are used initially, and two different nucleosides are used in step (e).