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 polynucleotide primer effective in reducing primer-dimer formation comprising at least one modified purine nucleobase comprising the structure
wherein R 1 is selected from hydrogen, azido, nitro, unsubstituted or substituted amino, C 1 -C 6 alkyl, and unsubstituted or substituted aryl,
at least one said modified purine nucleobase is no more than 3 nucleotides from the 3′ terminus of the primer, and the primer is extendable at its 3′-end, and
wherein the primer is effective in reducing primer-dimer formation.
2. The primer according to claim 1 , wherein at least one said modified purine nucleobase comprises the structure
3. The primer according to a claim 1 , wherein at least one said modified purine nucleobase comprises the structure
4. The primer according to claim 1 , wherein at least one said modified purine nucleobase comprises the structure
5. The primer according to claim 1 , wherein at least one said modified purine nucleobase is no more than 2 nucleotides from the 3′ terminus of the primer.
6. The primer according to claim 1 , wherein at least one said modified purine nucleobase is no more than 1 nucleotides from the 3′ terminus of the primer.
7. The primer according to claim 1 , wherein said modified purine nucleobase is the 3′ terminal nucleobase of the primer.
8. The primer according to claim 1 , comprising at least one of a detectable label, a quencher or a minor groove binder, or any combination thereof.
9. 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, wherein the polynucleotide primer is a primer according to claim 1 .
10. The method of claim 9 comprising, after the step of extending, denaturing the double stranded amplicon.
11. The method of claim 10 , wherein the steps of annealing, extending and denaturing are repeated at least one time.
12. The method of claim 10 , wherein the steps of annealing, extending and denaturing are repeated at least 10 times.
13. The method of claim 10 , wherein the steps of annealing, extending and denaturing are repeated at least 20 times.
14. The method of claim 10 , wherein the steps of annealing, extending and denaturing are repeated at least 30 times.
15. The method of claim 10 , wherein the steps of annealing, extending and denaturing are repeated at least 40 times.
16. The method according to claim 1 , wherein the extending takes place in the presence of extendable nucleotide triphosphates and non-extendable nucleotide triphosphates to form DNA amplicon fragments.
17. The method of claim 16 comprising, detecting the DNA amplicon fragments.