Modified nucleotides and methods of labeling nucleic acids

• US 20050003371A1
• Filed: 10/22/2003
• Published: 01/06/2005
• Est. Priority Date: 10/23/2002
• Status: Abandoned Application

• Current Assignee: Stratagene California
• Original Assignee: Stratagene California
• Inventors: Huang, Haoqiang, Anderson, Jack D., Braman, Jeffrey Carl
• Application Number:
  US10/691,269
• Publication Number:
  US 20050003371A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The invention relates to functionalized nucleotides that permit the covalent linkage of the nucleotides to moieties containing reactive groups complementary to the functional group. More specifically, the invention relates to nucleotides comprising a functional group attached to the nucleobase that permits the covalent attachment of the nucleotides or a nucleic acid comprising such nucleotides to detectable moieties, polypeptides and solid supports. The invention further relates to nucleic acids comprising such functionalized nucleotides, methods of labeling nucleic acids with such nucleotides, and kits comprising such nucleotides.

Claims

1. A nucleotide comprising the structure:

Phosphate-sugar-nucleobase-linker-F;

wherein F is a functional group selected from;

2. The nucleotide of claim 1 wherein said Linker is attached to said Nucleobase at the N-4 or C-5 position of said nucleobase when said nucleobase is a pyrimidine, or at the N-6, C-8 or C(N)-7 position of said nucleobase when said nucleobase is a purine.

3. The nucleotide of claim 1 wherein said nucleobase is selected from the group consisting of:

adenine, cytosine, guanine, thymine, uracil and hypoxanthine.

4. The nucleotide of claim 1 wherein said linker is selected from the group consisting of:

—

CH₂—

(CH₂—

CH₂)_v—

CH₂—

NHC(O)-Q-;

—

CH₂—

(CH₂—

CH₂)_V—

CH₂—

C(O)—

NH—

C(O)-Q-;

—

S—

CH₂C(O)-Q-;

—

S—

CH₂CH₂NH—

C(O)-Q-;

—

O—

CH₂C(O)-Q;

—

O—

CH₂CH₂NH—

C(O)-Q-;

—

NH—

(CH₂)_v—

NH—

C(O)-Q-;

v=0,1,2,3, Q=—

NH(CH₂)₆NH—

, —

NH—

(CH₂)₂—

NH, —

(CH₂)₅NH—

, —

(CH₂)₂—

C(O)—

NH—

(CH₂)₃—

O—

(CH₂)₂—

O—

(CH₂)₂—

O—

(CH₂)₃—

NH—

, —

NH—

[(CH₂)₂—

O—

)_w—

(CH₂)₂—

NH—

, —

(CH₂)₂ C(O)—

NH—

[(CH₂)₂—

O]_W—

NH—

, and w=2,3,4,5.

5. The nucleotide of claim 1 wherein said nucleotide is selected from the group consisting of ATP, dATP, ddATP, GTP, dGTP, ddGTP, CTP, dCTP, ddCTP, UTP, dUTP, TTP and ddTTP.

6. The nucleotide of claim 1 wherein said phosphate moiety is a mono-, di-, tri-, or tetraphosphate group.

7. The nucleotide of claim 1 wherein said sugar moiety is a cyclic pyranofuranose sugar.

8. The nucleotide of claim 7 wherein said cyclic pyranofuranose sugar is selected from the group consisting of ribofuranosyl, 2′

-deoxyribofuranosyl, and 2′

,3′

-dideoxyribofuranosyl.

9. The nucleotide of claim 1 wherein said sugar moiety is a cyclic non-furanose sugar.

10. The nucleotide of claim 9 wherein said cyclic non-furanose sugar is selected from the group consisting of oxetan, pyran or oxadiazepine.

11. The nucleotide of claim 1 wherein said sugar moiety is an acyclic sugar analog.

12. The nucleotide of claim 11 wherein said acyclic sugar analog is selected from the group consisting of phosphonomethoxyethyl, 2-oxyethoxymethyl, 2-hydroxymethoxymethyl, and 3-pentenyl.

13. A method of labeling a nucleotide of claim 1, said method comprising contacting said nucleotide with a detectable moiety comprising a reactive thiol group.

14. The method of claim 13 wherein said detectable moiety comprises a chromogenic dye, a fluorescent dye, a polypeptide or an enzyme.

15. A nucleotide labeled according to the method of claim 13.

16. A method of labeling a nucleic acid, said method comprising contacting said nucleic acid with a nucleotide of claim 15.

17. The method of claim 16 wherein said contacting is performed in the presence of a nucleic acid polymerase.

18. A method of labeling a nucleic acid, said method comprising contacting said nucleic acid with a nucleotide of claim 1.

19. The method of claim 18 wherein said contacting is performed in the presence of a nucleic acid polymerase.

20. The method of claim 18 further comprising contacting said nucleotide with a thiol-containing detectable moiety.

21. The method of claim 20, wherein said thiol-containing detectable moiety is a chromogenic moiety, a fluorescent dye, a polypeptide or an enzyme.

22. A polynucleotide comprising a nucleotide of claim 1.

23. A method of attaching a nucleic acid to a surface, said method comprising:

a) contacting said nucleic acid with a nucleotide of claim 1 in the presence of a nucleic acid polymerase, wherein said contacting results in the incorporation of said nucleotide into said nucleic acid or its complement;

b) contacting the nucleic acid of step (a) with a surface comprising a reactive group complementary to the functional group F on said nucleotide, wherein said contacting results in covalent attachment of said nucleic acid of step (a) to said surface.

24. The method of claim 23 wherein said surface is a plate, tube, bead or column matrix.

25. A kit comprising a nucleotide of claim 1.

26. The kit of claim 25, further comprising a nucleic acid polymerase, and packaging materials therefor.

27. A nucleotide comprising the structure:

Phosphate-Sugar-Nucleobase-F

wherein F is a functional group selected from;

wherein said sugar is an acyclic sugar analog.

28. The nucleotide of claim 27 wherein said acyclic sugar analog is selected from the group consisting of phosphonomethoxyethyl, 2-oxyethoxymethyl, 2-hydroxymethoxymethyl, and 3-pentenyl.

29. A polynucleotide comprising a nucleotide of claim 27.

30. A kit comprising a nucleotide of claim 27.

31. The kit of claim 30, further comprising a nucleic acid polymerase, and packaging materials therefor.