Compositions and methods for nucleotide sequencing

• US 9,469,873 B2
• Filed: 04/24/2015
• Issued: 10/18/2016
• 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:
  US14/695,955
• Publication Number:
  US 20150225785A1
• Time in Patent Office: 543 Days
• Field of Search: 536/4.1, 536/23.1, 536/26.6, 435/6.12
• 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 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 4′

position of said sugar moiety, is a blocking group comprising a phosphate.

2. The nucleotide of claim 1, wherein said label is a fluorescent dye.

3. The nucleotide of claim 1, wherein said label comprises a ligand.

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

5. The nucleotide of claim 1, wherein said blocking group is cleavable by enzymatic, photolytic and/or chemical cleavage.

6. The nucleotide of claim 1, wherein the sugar moiety further comprises a blocking group attached at the 2′

position.

7. The nucleotide of claim 1, wherein the linker comprises an enzymatically cleavable peptide linker.

8. The nucleotide of claim 7, wherein said enzymatically cleavable peptide linker can be cleaved by a protease.

9. A nucleic acid molecule comprising the nucleotide of claim 1.

10. The nucleic acid molecule of claim 9, wherein said nucleotide is at the 5′

or 3′

terminus of the nucleic acid.

11. A kit comprising the nucleotide of claim 1 and polymerase configured to incorporate the nucleotide into a growing nucleic acid molecule.

12. The kit of claim 11, wherein linker comprises an enzymatically cleavable peptide linker and wherein the kit further comprises an enzyme configured to cleave the linker.

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

wherein, attached at the 4′

position of said sugar moiety, is a 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; and

wherein the linker comprises an enzymatically cleavable peptide linker.

14. The nucleotide of claim 13, wherein said blocking group comprises a phosphate.

15. The nucleotide of claim 13, wherein said label is a fluorescent dye.

16. The nucleotide of claim 13, wherein said label comprises a ligand.

17. The nucleotide of claim 13, wherein said nucleotide molecule is a nucleotide triphosphate.

18. The nucleotide of claim 13, wherein said blocking group is cleavable by enzymatic, photolytic and/or chemical cleavage.

19. The nucleotide of claim 13, wherein the sugar moiety further comprises a blocking group attached at the 2′

position.

20. A nucleic acid molecule comprising the nucleotide of claim 13.