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

• US 8,361,727 B2
• Filed: 05/24/2011
• Issued: 01/29/2013
• Est. Priority Date: 12/05/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Wu, Weidong, Litosh, Vladislav A., Stupi, Brian P., Metzker, Michael L.
• Primary Examiner(s): Staples, Mark
• Application Number:
  US13/114,270
• Publication Number:
  US 20110287427A1
• Time in Patent Office: 616 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 method of sequencing a target nucleic acid comprising the following steps:

(i) attaching the 5′

-end of a nucleic acid to a solid surface;

(ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a hybridized primer/target nucleic acid complex;

(iii) obtaining a polymerase and one or more compounds according to formula I, II, III, IV, V, VI or VII;

2. The method according to claim 1, wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the efficiency of incorporation of its natural nucleotide counterpart.

3. The method according to claim 2, wherein the incorporation efficiency occurs at about 85% to about 100%.

4. The method according to claim 1, wherein the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and DNA polymerase.

5. The method according to claim 1, wherein about 85% to about 100% of the photocleavable terminating moieties are removed by exposure to a light source in step (vi).

6. The method according to claim 1, wherein incorporation of at least one compound according to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.

7. The method of claim 1, wherein a pulsed multiline excitation detector is used for imaging in step (v).

8. The method of claim 1, further comprising washing the growing primer strand after step (iv).

9. The method of claim 1, further comprising washing the extended primer after step (vi).

10. The method of claim 1, further comprising prior to step (iv) capping any primers or growing primer strands that did not react in step (iv).

11. The method of 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.

12. The method of claim 1, wherein R₃ and 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.

13. The method of claim 1, wherein R₅, R₇, and R₈ are each H.

14. The method of claim 1, wherein R₃ or R₄ is —

CH₃ or isopropyl.

15. The method of claim 1, where the compound is further defined as:

16. The method of claim 1, where the compound is further defined as:

17. The method of claim 1, where the compound is further defined as:

18. The method of claim 1, where the compound is further defined as:

19. The method of claim 1, where the compound is further defined as:

20. The method of claim 1, where the compound is further defined as:

21. The method of claim 1, where the compound is further defined as:

22. The method of claim 1, where the compound is further defined as:

23. The method of claim 1, where the compound is further defined as:

24. The method of claim 1, where the compound is further defined as:

25. The method of claim 1, where the compound is further defined as:

26. The method of claim 1, where the compound is further defined as:

27. The method of claim 1, further comprising sequencing multiple target nucleic acids synchronistically.

28. The method of claim 1, where the compound is further defined as a compound of formula (I).

29. The method of claim 1, where the compound is further defined as a compound of formula (II).

30. The method of claim 1, where the compound is further defined as a compound of formula (III).

31. The method of claim 1, where the compound is further defined as a compound of formula (IV).

32. The method of claim 1, where the compound is further defined as a compound of formula (V).

33. The method of claim 1, where the compound is further defined as a compound of formula (VI).

34. The method of claim 1, where the compound is further defined as a compound of formula (VII).