Modified oligonucleotides and applications thereof
View Patent ↗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 method of primer extension comprising,
i) annealing a polynucleotide primer to a denatured DNA template such that, the polynucleotide primer anneals to a complementary polynucleotide sequence on a strand of the denatured DNA template to form a primer-template complex, and
ii) extending the primer portion of the primer-template complex to form a double stranded amplicon, and
wherein the polynucleotide primer comprises at least one modified pyrimidine nucleobase of the structure
wherein
X is N or C,
R 1 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently —H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl, and
R 2 is selected from —H, —F, —Cl, —Br, C 1 -C 6 alkyl, C 1 -C 6 substituted alkyl, C 3 -C 10 aryl, C 3 -C 10 substituted aryl, —CF 3 , —CF 2 H, —CF 2 CH 3 , —CF 2 CF 3 , —CCl 3 , —CN, —CHO, —CO 2 R, —SO 3 R, —PO 3 RR, —C(O)NRR, azido, and —NO 2 where each R is independently —H, C 1 -C 6 alkyl or C 3 -C 10 aryl or alkylaryl,
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 primer.
2. The method of claim 1 comprising, after the step of extending, denaturing the double stranded amplicon.
3. The method of claim 2 , wherein the steps of annealing, extending and denaturing are repeated at least one time.
4. The method of claim 2 , wherein the steps of annealing, extending and denaturing are repeated at least 10 times.
5. The method of claim 2 , wherein the steps of annealing, extending and denaturing are repeated at least 20 times.
6. The method of claim 2 , wherein the steps of annealing, extending and denaturing are repeated at least 30 times.
7. The method of claim 2 , wherein the steps of annealing, extending and denaturing are repeated at least 40 times.
8. The method according to claim 3 , wherein the extending takes place in the presence of extendable nucleotide triphosphates and non-extendable nucleotide triphosphates to form DNA amplicon fragments.
9. The method of claim 8 comprising, detecting the DNA amplicon fragments.
10. A method of primer extension comprising:
i) annealing a first polynucleotide primer and a second polynucleotide primer to a first and second strand of a denatured DNA template such that, the first polynucleotide primer anneals to a complementary oligonucleotide sequence on the first strand of the denatured DNA template and the second polynucleotide primer anneals to a complementary oligonucleotide sequence on the second strand of the denatured DNA template to form a first and a second primer-template complex, and
ii) extending the primer portion of at least one of the first and second primer-template complex to form double stranded DNA amplicon,
wherein at least one of the first polynucleotide primer or the second polynucleotide primer is comprises at least one modified pyrimidine nucleobase according to claim 1 .
11. The method of claim 10 comprising after the step of forming but prior to the step of annealing, denaturing the DNA template to form a first strand of denatured DNA template and a second denatured DNA template.
12. The method according to claim 11 comprising, after the step of extending, denaturing the double stranded DNA amplicon.
13. The method of claim 12 wherein the steps of annealing, extending and denaturing the double stranded DNA amplicon are repeated at least 1 time.
14. The method of claim 12 wherein the steps of annealing, extending and denaturing the double stranded DNA amplicon are repeated at least 10 times.
15. The method of claim 12 wherein the steps of annealing, extending and denaturing the double stranded DNA amplicon are repeated at least 20 times.
16. The method of claim 12 wherein the steps of annealing, extending and denaturing the double stranded DNA amplicon are repeated at least 30 times.
17. The method according to claim 10 comprising prior to the step of extending the primer portion, annealing a polynucleotide probe to one of a first or second strand of a denatured DNA template such that, the polynucleotide probe anneals to a complementary polynucleotide sequence on the first strand of the denatured DNA template or the polynucleotide probe anneals to a complementary oligonucleotide sequence on the second strand of the denatured DNA template.
18. The method of claim 17 , wherein the polynucleotide probe comprises at least one detectable label.
19. The method of claim 18 , wherein the polynucleotide probe further comprises at least one of a quencher, a minor groove binder or both.
20. The method of claim 1 , wherein the at least one modified pyrimidine nucleobase of the polynucleotide primer is the 3′ terminal nucleotide.
21. The method of claim 1 , wherein at least one modified pyrimidine nucleobase is no more than 3 nucleotides from the 3′ terminus of the polynucleotide.
22. The method of claim 1 , wherein at least one modified pyrimidine nucleobase is no more than 2 nucleotides from the 3′ terminus of the polynucleotide.
23. The method of claim 1 , wherein the polynucleotide primer is extendable at the 3′-terminus.
24. The method of claim 1 , wherein the polynucleotide primer is a probe that is not extendable at its 3′ terminus.