Nucleotide compounds including a rigid linker

• US 5,948,648 A
• Filed: 05/29/1998
• Issued: 09/07/1999
• Est. Priority Date: 05/29/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems Division Perkin-Elmer Corporation
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B., Zhen, Weiguo
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US09/087,250
• Time in Patent Office: 466 Days
• Field of Search: 536/23.1, 536/22.1, 536/24.3, 536/25.3, 536/25.32, 536/25.33, 536/25.34, 536/26.21, 536/26.6, 536/26.8, 536/27.21, 536/27.6, 536/27.8, 536/27.81 , 536/28.1, 536/28.2, 536/28.5, 536/28.53, 536/28.54, 435/87, 435/88, 435/89, 435/91.1
• US Class Current: 435/87
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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

Abstract

A nucleoside/tide compound having the structure

NUC--L--S--LB/LG

is described wherein NUC is a nucleoside/tide having a nucleobase portion B, L is a rigid linkage, S is a spacer; and LB/LG is a member of a linkage pair or a label. NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine. In an important aspect of the invention, L has the structure ##STR1## wherein each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2, and each of W, X, Y and Z is selected from the group consisting of carbon and nitrogen. The invention further includes polynucleotide compounds comprising the nuclcoside/tide, and primer extension methods utilizing the nucleoside/tide, particularly when used in combination with certain mutant polymerase enzymes.

Claims

1. A nucleoside/tide compound having the structure

View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

2. The compound of claim 1 wherein NUC is selected from the group consisting of a substituent radical derived from a 2'"'"'-deoxyribonucleotide, a 3'"'"' deoxyribonucleotide, a 2'"'"',3'"'"'-dideoxyribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-fluoro-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-fluoro-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-azido-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-azido-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-amino-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-amino-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"',3'"'"' didehydroribonucleotide and a ribonucleotide.

3. The compound of claim 2 wherein NUC is selected from the group consisting of a substituent radical derived from a 2'"'"',3'"'"'-dideoxyribonucleotide and a 2'"'"',3'"'"'-fluoro-ribonucleotide.

4. The compound of claim 1 wherein one of W and X is CH and one of Z and Y is CH.

5. The compound of claim 1 wherein n is 1 or 2.

6. The compound of claim 1 wherein o is 1 or 2.

7. The compound of claim 1 wherein p is 0 or 1.

8. The compound of claim 1 wherein n is 1 or 2, o is 1 or 2, and p is 0 or 1.

9. The compound of claim 1 wherein S has the structure ##STR11## where n ranges from 1 to 8.

10. The compound of claim 9 wherein n is 1 or 2.

11. The compound of claim 1 wherein LB/LG is a member of a linkage pair.

12. The compound of claim 11 wherein the member of the linkage pair is amine.

13. The compound of claim 12 wherein the amine is a primary amine.

14. The compound of claim 1 wherein LB/LG is a label.

15. The compound of claim 14 wherein the label is a xanthene-type dye.

16. The compound of claim 15 wherein the xanthene-type dye is a rhodamine or a fluorescein dye.

17. The compound of claim 1 wherein --L-- has the structure ##STR12##

18. The compound of claim 1 wherein --L-- has the structure --C.tbd.C--C.tbd.C--.

19. The compound of claim 1 wherein --L-- has the structure

20. The compound of claim 1 wherein --L-- has the structure

21. The compound of claim 1 wherein --L-- has the structure

22. The compound of claim 1 wherein --L-- has the structure

23. A polynucleotide comprising a nucleotide compound having the structure

View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)

24. The compound of claim 23 wherein NUC is selected from the group consisting of a substituent radical derived from a 2'"'"'-deoxyribonucleotide, a 3'"'"'-deoxyribonucleotide, a 2'"'"',3'"'"'-dideoxyribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-fluoro-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-fluoro-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-azido-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-azido-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"'-amino-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-3'"'"'-amino-ribonucleotide, a 2'"'"',3'"'"'-dideoxy-2'"'"',3'"'"'didehydroribonucleotide and a ribonucleotide.

25. The compound of claim 23 wherein NUC is selected from the group consisting of a substituent radical derived from a 2'"'"',3'"'"'-dideoxyribonucleotide and a 2'"'"',3'"'"'-dideoxy-3'"'"'-fluoro-ribonucleotide.

26. The compound of claim 23 wherein one of W and X is CH and one of Z and Y is CH.

27. The compound of claim 23 wherein n is 1 or 2.

28. The compound of claim 23 wherein o is 1 or 2.

29. The compound of claim 23 wherein p is 0 or 1.

30. The compound of claim 23 wherein n is 1 or 2, o is 1 or 2, and p is 0 or 1.

31. The compound of claim 23 wherein S has the structure ##STR14## where n ranges from 1 to 8.

32. The compound of claim 31 wherein n is 1 or 2.

33. The compound of claim 23 wherein LB/LG is a member of a linkage pair.

34. The compound of claim 33 wherein the member of the linkage pair is amino.

35. The compound of claim 34 wherein the amino is a primary amine.

36. The compound of claim 23 wherein LB/LG is a label.

37. The compound of claim 36 wherein the label is a xanthene-type dye.

38. The compound of claim 37 wherein the xanthene-type dye is a rhodamine or a fluorescein dye.

39. A method for performing a primer extension reaction comprising the steps of:

providing a template nucleic acid;

annealing an oligonucleotide primer to a portion of the template nucleic acid for forming a primer-template hybrid; and

adding primer-extension reagents to the primer-template hybrid for extending the primer, the primer extension reagents including a nucleotide compound having the structure

space="preserve" listing-type="equation">NUC--L--S--LB/LG

wherein;

NUC is a nucleoside/tide having a nucleobase portion B;

L is a rigid linkage;

S is a spacer; and

LB/LG is a member of a linkage pair or a label;

wherein

NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine; and

L has the structure ##STR15## wherein each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2; and

each of W, X, Y and Z is selected from the group consisting of CH and N.

40. The method of claim 39 wherein the primer extension reagent includes a theromostable DNA polymerase.

41. The method of claim 40 wherein the thermostable polymerase is a Taq polymerase or a mutant thereof.

42. The method of claim 41 wherein the Taq polymerase is a mutant Taq polymerase having a mutation at the F667 position.

43. The method of claim 41 wherein the Taq polymerase is a mutant Taq polymerase having a mutation at the R660 position.

44. The method of claim 43 wherein the mutation is selected from the group consisting of R660C, R660D and R660S.