Modified oligonucleotides and applications thereof
Disclosed, among other things, are primers containing certain modified nucleobases in the 3′ terminal region of the primers that provide reduced formation of primer-dimers during amplification reactions, and various methods of use thereof.
1. A polynucleotide comprising:
at least one modified pyrimidine nucleobase of the structure:
wherein X is N or C;
wherein at least one of R 1 or R 2 is an electron withdrawing substituent or X is N, such that when X is N, R 2 is absent; and
at least one said modified pyrimidine nucleobase is no more than 4 nucleotides from the 3′ terminus of the polynucleotide, the polynucleotide operable to reduce primer-dimer formation in nucleic acid amplification reactions.
2. The polynucleotide according to claim 1 , wherein R 1 is —CN.
3. The polynucleotide according to claim 1 , wherein R 1 is —F.
4. The polynucleotide according to claim 1 , wherein R 1 is —CF 3 .
5. The polynucleotide according to claim 1 , wherein R 2 is —CN.
6. The polynucleotide according to claim 1 , wherein R 2 is —F.
7. The polynucleotide according to claim 1 , wherein R 2 is —CF 3 .
8. The polynucleotide according to claim 1 , wherein X is C.
9. The polynucleotide according to claim 8 , wherein R 2 is —H.
10. The polynucleotide according to claim 9 , wherein R 1 is —CN.
11. The polynucleotide according to claim 9 , wherein R 1 is —F.
12. The polynucleotide according to claim 9 , wherein R 1 is —CF 3 .
13. The polynucleotide according to claim 8 , wherein R 2 is —CN.
14. The polynucleotide according to claim 8 , wherein R 2 is —F.
15. The polynucleotide according to claim 8 , wherein R 2 is —CF 3 .
16. The polynucleotide according to claim 1 , wherein X is N.
17. The polynucleotide according to claim 16 , wherein R 1 is —CN.
18. The polynucleotide according to claim 16 , wherein R 1 is —F.
19. The polynucleotide according to claim 16 , wherein R 1 is —CF 3 .
20. A method for reducing formation of primer-dimers comprising:
amplifying a target nucleic acid using at least two primers having at least one modified pyrimidine nucleobase having a structure comprising:
wherein X is N or C;
wherein at least one of R 1 or R 2 is an electron withdrawing substituent or X is N, such that when X is N, R 2 is absent; and
at least one said modified pyrimidine nucleobase is no more than 4 nucleotides from the 3′ terminus of the polynucleotide, the polynucleotide operable to reduce primer-dimer formation in nucleic acid amplification reactions.
21. The method of claim 20 , wherein the at least one modified pyrimidine nucleobase has a structure comprising:
22. The method of claim 20 , wherein the at least one modified pyrimidine nucleobase has a structure comprising:
23. The method of claim 20 , wherein the at least one modified pyrimidine nucleobase has a structure comprising: