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 method of DNA amplicon fragment analysis comprising:
i) annealing an oligonucleotide primer to a denatured DNA template such that, the oligonucleotide primer anneals to a complementary oligonucleotide sequence on a strand of the denatured DNA template to form a primer-template complex, wherein the oligonucleotide primer comprises at least one modified purine nucleobase comprising the structure:
wherein R 1 is selected from hydrogen, halogen, fluorine, chlorine, bromine, iodine, azido, nitro, cyano, unsubstituted or substituted amino, C 1 -C 6 alkyl, C 1 -C 6 alkynyl, C 1 -C 6 substituted alkynyl, unsubstituted or substituted phenylalkynyl, and unsubstituted or substituted aryl;
at least one said modified purine nucleobase is no more than 3 nucleotides from the 3′ terminus of the oligonucleotide primer; and
the oligonucleotide primer is extendable at its 3′-end;
ii) extending the primer portion of the primer-template complex in the presence of extendable nucleotide triphosphates and non-extendable nucleotide triphosphates to form one or more DNA amplicon fragments; and
iii) detecting the DNA amplicon fragments,
wherein the oligonucleotide primer comprising at least one modified purine nucleobase no more than 3 nucleotides from the 3′ terminus of the oligonucleotide primer significantly reduces the formation of primer-dimer amplicons.
2. The method of claim 1 , wherein the at least one modified purine nucleobase is no more than 2 nucleotides from the 3′ terminus of the oligonucleotide primer.
3. The method of claim 1 , wherein the at least one modified purine nucleobase is no more than 1 nucleotides from the 3′ terminus of the oligonucleotide primer.
4. The method of claim 1 , wherein the at least one modified purine nucleobase is the 3′ terminal nucleotide of the oligonucleotide primer.
5. The method of claim 1 , wherein detecting the amplicon DNA fragments comprises a size-dependent separation process to generate separated DNA amplicon fragments.
6. The method of claim 5 wherein the size dependent separation process may comprise electrophoresis, chromatography or combinations thereof.
7. The method of claim 1 , wherein the oligonucleotide primer further comprises at least one of a detectable label, a quencher or a minor groove binder, or any combination thereof.
8. The method of claim 7 , comprising generating labeled DNA amplicon fragments.
9. The method of claim 8 further comprising detecting the DNA amplicon fragments by laser-induced fluorescence.
10. The method of claim 7 , comprising using a plurality of labeled oligonucleotide primers wherein each labeled oligonucleotide primer is labeled with a different and spectrally resolvable label.
11. The method of claim 10 , wherein the DNA amlicon fragments are formed using chain termination methods of DNA sequencing.
12. The method of claim 11 wherein the DNA amplicon fragments may be identified by identifying terminal nucleotides in each of the DNA amplicon fragment and wherein a correspondence is established between the four possible terminal nucleotides and a set of spectrally resolvable labels.
13. The method of claim 1 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure:
14. The method of claim 1 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure:
15. The method of claim 1 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure:
16. A method of DNA amplicon fragment analysis comprising:
i) annealing an oligonucleotide primer to a denatured DNA template such that, the oligonucleotide primer anneals to a complementary oligonucleotide sequence on a strand of the denatured DNA template to form a primer-template complex, wherein the oligonucleotide primer comprises at least one modified purine nucleobase comprising the structure:
wherein R 1 is selected from hydrogen, halogen, fluorine, chlorine, bromine, iodine, azido, nitro, cyano, unsubstituted or substituted amino, C 1 -C 6 alkyl, C 1 -C 6 alkynyl, C 1 -C 6 substituted alkynyl, unsubstituted or substituted phenylalkynyl, and unsubstituted or substituted aryl;
at least one said modified purine nucleobase is no more than 3 nucleotides from the 3′ terminus of the oligonucleotide primer, and
the oligonucleotide primer is extendable at its 3′-end;
ii) extending the primer portion of the primer-template complex in the presence of deoxyribonucleic acids and non-extendable ribonucleic acids to form one or more DNA amplicon fragments; and
iii) detecting the DNA amplicon fragments,
wherein the oligonucleotide primer comprising at least one modified purine nucleobase no more than 3 nucleotides from the 3′ terminus of the oligonucleotide primer significantly reduces the formation of primer-dimer amplicons.
17. The method of claim 16 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure:
18. The method of claim 16 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure:
19. The method of claim 16 , wherein the oligonucleotide primer comprising the at least one said modified purine nucleobase comprises the structure: