3′-OH unblocked, nucleotides and nucleosides, base modified with photocleavable, terminating groups and methods for their use in DNA sequencing

• US 7,897,737 B2
• Filed: 12/05/2006
• Issued: 03/01/2011
• Est. Priority Date: 12/05/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Stupi, Brian P., Wu, Weidong, Metzker, Michael L., Litosh, Vladislav A.
• Primary Examiner(s): Staples; Mark
• Application Number:
  US11/567,189
• Publication Number:
  US 20080132692A1
• Time in Patent Office: 1,547 Days
• Field of Search: None
• US Class Current: 536/22.1
• CPC Class Codes: C07H 17/02 Heterocyclic radicals containing only nitrogen as ring hetero atoms
• 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 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a photocleavable terminating group. The photocleavable-fluorescent group is designed to terminate DNA synthesis as well as be cleaved so that DNA oligomers can be sequenced efficiently in a parallel format. The design of such rapidly cleavable fluorescent groups on nucleotides and nucleosides can enhance the speed and accuracy of sequencing of large oligomers of DNA in parallel, to allow rapid whole genome sequencing, and the identification of polymorphisms and other valuable genetic information, as well as allowing further manipulation and analysis of nucleic acid molecules in their native state following cleavage of the fluorescent group.

Claims

1. A compound selected from the group consisting of:

2. A compound according to claim 1, wherein R₃ and R₄ are each independently selected from the group consisting of H, —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃, isopropyl, tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl, with the proviso that at least one of R₃ and R₄ is H.

3. A compound according to claim 1, wherein R₃ or R₄ are each independently selected from the group consisting of H, alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group, with the proviso that at least one of R₃ and R₄ is H.

4. The compound of claim 2, wherein R₃ or R₄ is —

CH₃ or isopropyl.

5. The compound of claim 1, further defined as:

6. The compound of claim 5, further defined as:

7. The compound of claim 1, further defined as:

8. The compound of claim 1, further defined as:

9. The compound of claim 1, further defined as:

10. The compound of claim 1, further defined as:

11. The compound of claim 1, further defined as:

12. The compound of claim 1, further defined as:

13. The compound of claim 1, further defined as:

14. The compound of claim 1, further defined as:

15. The compound of claim 1, further defined as:

16. The compound of claim 1, further defined as:

17. The compound of claim 1, further defined as:

18. The compound of claim 1, further defined as:

19. The compound of claim 1, further defined as a compound of formula (I).

20. The compound of claim 1, further defined as a compound of formula (II).

21. The compound of claim 1, further defined as a compound of formula (III).

22. The compound of claim 1, further defined as a compound of formula (IV).

23. The compound of claim 1, further defined as a compound of formula (V).

24. The compound of claim 1, further defined as a compound of formula (VI).

25. The compound of claim 1, further defined as a compound of formula (VII).

26. A compound according to any one of claims 19-25, wherein R₃ and R₄ are each independently selected from the group consisting of H, —

CH₃, —

CH₂CH₃, —

CH₂CH₂CH₃, isopropyl, tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl, with the proviso that at least one of R₃ and R₄ is H.

27. A compound according to any one of claims 19-25, wherein R₃ or R₄ are each independently selected from the group consisting of H, alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group, with the proviso that at least one of R₃ and R₄ is H.

28. A compound according to any one of claims 19-25, wherein R₃ or R₄ is —

CH₃ or isopropyl.