Compositions and methods for nucleotide sequencing

• US 9,051,612 B2
• Filed: 07/10/2014
• Issued: 06/09/2015
• Est. Priority Date: 09/28/2006
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Zhao, Chanfeng, Kozlov, Igor, Won, Melissa
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/327,872
• Publication Number:
  US 20140370498A1
• Time in Patent Office: 334 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The invention provides nucleoside and nucleotide molecules containing cleavable linkers linking a label such as a dye. The invention also provides nucleosides and nucleotide molecules containing a blocking group, either removable or non-removable. The invention additionally provides methods of using the nucleoside and nucleotide molecules containing a cleavable linker and/or a blocking group.

Claims

1. A method for sequencing a nucleic acid molecule, comprising:

(a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, an extension enzyme, and a reversibly blocked nucleotide under conditions wherein said primer is extended to incorporate said nucleotide into a sequence complementary to said nucleic acid template,

said reversibly blocked nucleotide comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by linker, and

wherein, attached at the position of said sugar moiety, is a reversible blocking group comprising —

CH₂—

CH═

CH₂, —

CH₂—

N₃, —

CH₂—

S—

S-tBu, —

CH₂—

S-Me, —

CH₂—

O—

CH₂—

CH₂—

CN, —

CH₂—

CH₂—

CN, —

C(O)—

CH₃, —

CH₃, 2-nitrobenzyl, tetrahydropyran, 4-nitrobenzoyl, 2-aminobenzoyl, —

C(O)-linker-dye, 2-nitrobenzyl-linker-dye, —

C(O)—

O—

CH₂—

CH═

CH-linker-dye, —

NH—

C(O)—

CH₃, —

F, —

NH₂, phosphate, or sulfate,

(b) identifying the nucleotide in said sequence complementary to said nucleic acid template;

(c1) removing or modifying the label; and

(c2) removing the blocking group,

wherein identifying said nucleotide identifies at least a portion of the sequence of said nucleic acid molecule.

2. The method of claim 1, wherein step (c1) comprises reducing a signal from the label by chemical quenching.

3. The method of claim 1, wherein step (c1) comprises reducing a signal from the label by thermal quenching.

4. The method of claim 1, wherein step (c1) comprises reducing a signal from the label by photobleaching.

5. The method of claim 1, wherein step (c1) comprises removing or modifying the label while leaving the linker intact.

6. The method of claim 1, wherein step (c1) comprises chemically or enzymatically cleaving the linker.

7. The method of claim 1, wherein said blocking group comprises a phosphate.

8. The method of claim 1, wherein said label is a fluorescent dye.

9. The method of claim 1, wherein said label comprises a ligand.

10. The method of claim 1, wherein said nucleotide molecule is a nucleotide triphosphate.

11. The method of claim 1, wherein step (c2) comprises enzymatically, photolytically and/or chemically cleaving said blocking group.

12. The method of claim 1, further comprising adding a second nucleotide and repeating step (a), wherein said complementary sequence is extended and said second nucleotide is incorporated into said sequence complementary to said nucleic acid template.

13. The method of claim 7, further comprising repeating step (b), wherein identifying said second nucleotide identifies at least a portion of the sequence of said nucleic acid template.

14. The method of claim 8, further comprising repeating steps (c1) and (c2).

15. The method of claim 6, wherein steps (c1) and (c2) are carried out in the same reaction.

16. The method of claim 6, further comprising adding one or more nucleotides and repeating steps (a), (b), (c1) and (c2) one or more times, wherein at least a portion of the sequence of said nucleic acid template is determined.

17. The method of claim 1, wherein step (a) is performed by providing an extension enzyme selected from the group consisting of a polymerase, terminal transferase, reverse transcriptase, polynucleotide phosphorylase, and telomerase.

18. The method of claim 1, wherein said nucleic acid molecule comprises a primer and said primer is hybridized to a template nucleic acid.

19. The method of claim 1, wherein the sugar moiety has a blocking group attached at the 2′

position.