COMPOSITIONS AND METHODS FOR NUCLEOTIDE SEQUENCING

• US 20100092957A1
• Filed: 09/04/2007
• Published: 04/15/2010
• Est. Priority Date: 09/28/2006
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Shults, Melissa, Kozlov, Igor, Zhao, Chanfeng
• Granted Patent: US 8,399,188 B2
• Time in Patent Office: Days
• US Class Current: 435/6.13
• 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 nucleoside, comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′

or 4′

position of said sugar moiety.

2. The molecule of claim 1, wherein said blocking group is removable.

3. The molecule of claim 1, wherein said label is a fluorescent dye.

4. The molecule of claim 1, wherein said linker can be cleaved by a protease.

5. The molecule of claim 1, wherein said label comprises a ligand.

6. A nucleotide molecule comprising the nucleoside of claim 1.

7. The nucleotide molecule of claim 6, wherein said nucleotide molecule is a nucleotide triphosphate.

8. A nucleic acid molecule comprising the nucleoside of claim 1.

9. The nucleic acid molecule of claim 8, wherein said nucleoside is at the 5′

or 3′

terminus of the nucleic acid.

10. 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, a polymerase and an RT-NTP under conditions wherein said primer is extended to incorporate said RT-NTP into a sequence complementary to said nucleic acid template, wherein said RT-NTP comprises a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′

or 4′

position of said sugar moiety; and

(b) identifying the RT-NTP in said sequence complementary to said nucleic acid template, wherein identifying said RT-NTP identifies at least a portion of the sequence of said nucleic acid molecule.

11. The method of claim 10, further comprising (c) removing said removable blocking group from said RT-NTP in said sequence complementary to said nucleic acid template.

12. The method of claim 11, wherein said removable blocking group is removed by an enzyme.

13. The method of claim 11, further comprising adding a second RT-NTP and repeating step (a), wherein said complementary sequence is extended and said second RT-NTP is incorporated into said sequence complementary to said nucleic acid template.

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

15. The method of claim 14, further comprising repeating step (c).

16. The method of claim 11, further comprising (d) cleaving said cleavable linker to remove said label from said RT-NTP in said sequence complementary to said nucleic acid template.

17. The method of claim 16, wherein steps (c) and (d) are carried out in the same reaction.

18. The method of claim 16, further comprising adding one or more RT-NTPs and repeating steps (a), (b), (c) and (d) one or more times, wherein at least a portion of the sequence of said nucleic acid template is determined.

19. A method for extending a primer nucleic acid, comprising contacting a nucleic acid molecule with a nucleotide incorporating catalyst and a nucleotide molecule, wherein said nucleotide molecule comprises a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′

or 4′

position of said sugar moiety, whereby said catalyst extends the nucleic acid to produce an extended nucleic acid molecule incorporating said nucleotide molecule.

20. The method of claim 19, further comprising removing said label and said blocking group from said nucleotide using enzymes.

21. The method of claim 20, wherein said label and said blocking group are removed using different enzymes contacted with the nucleotide in the same reaction.

22. The method of claim 19, wherein said catalyst is selected from a polymerase, terminal transferase, reverse transcriptase, polynucleotide phosphorylase and telomerase.

23. The method of claim 19, wherein said nucleic acid molecule comprises a primer and said primer is hybridized to a template nucleic acid.

24. A method of ligating two nucleic acid molecules, comprising incubating first and second nucleic acid molecules in the presence of a ligase, wherein at least one of said first and second nucleic acid molecules comprises on the 3′

end a nucleoside comprising a removable blocking group, wherein said removable blocking group is attached at the 2′

or 4′

position of the sugar moiety of said nucleoside.

25. The method of claim 24, wherein said nucleoside further comprises a label linked by an enzymatically cleavable peptide linker.

26. A kit comprising a nucleotide molecule and a nucleotide incorporating catalyst, wherein said nucleotide molecule comprises a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker and wherein a blocking group is attached at the 2′

or 4′

position of said sugar moiety, wherein said polymerase is capable of extending a primer nucleic acid to produce an extended primer nucleic acid molecule incorporating said nucleotide molecule.

27. The kit of claim 26, wherein said blocking group is enzymatically removable.

28. The kit of claim 27, further comprising an enzyme capable of removing said blocking group.

29. The kit of claim 26, further comprising a protease having activity to cleave said peptide linker.

30. The kit of claim 26, wherein said catalyst is selected from a polymerase, terminal transferase, reverse transcriptase, polynucleotide phosphorylase and telomerase.

31. A nucleoside, comprising a sugar moiety having a blocking group attached at the 2′

or 4′

position of said sugar moiety, wherein said blocking group comprises —

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, tetrandyropyran, 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.

32. A nucleoside, comprising a base moiety and a label linked to said base moiety by a peptide linker, wherein said linker can be cleaved by a protease selected from the group consisting of proteinase K, trypsin, chymotripsin, subtilisin and V8 protease.