Compositions of toehold primer duplexes and methods of use
Provided herein are primers and primer systems having improved specificity and kinetics over existing primers, and methods of use thereof.
1. A single-stranded primer that partially self-hybridizes to form
(1) a double-stranded target-non-specific region,
(2) a double-stranded target-specific region,
(3) a single-stranded target-specific region, and
(4) a hairpin loop region,
wherein the double-stranded target-non-specific region has a concentration-adjusted standard free energy that is within 10% of the concentration-adjusted standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
2. The single-stranded primer of claim 1 , wherein the double-stranded target non-specific region is about 4-21 nucleotide base pairs in length.
3. The single-stranded primer of claim 1 , wherein the single-stranded target-specific region is about 4-20 nucleotides in length.
4. The single-stranded primer of claim 1 , wherein the hairpin loop region is 4-30 nucleotides in length.
5. The single-stranded primer of claim 1 , wherein the single-stranded primer is comprised of deoxyribonucleic acid (DNA).
6. The single-stranded primer of claim 1 , wherein the single-stranded primer is comprised of ribonucleic acid (RNA).
7. A method of performing a multiplexed nucleic acid amplification reaction comprising amplifying multiple unique nucleic acid molecules using the primer of claim 1 .
8. A method comprising
contacting the single-stranded primer of claim 1 to a sample, and detecting hybridization of the primer to a target in the sample.
9. A method comprising performing a nucleic acid synthesis reaction in the presence of a nucleic acid target, a polymerase, and at least one single-stranded primer of claim 1 .
10. A kit comprising
at least one single-stranded primer of claim 1 , and
at least one nucleic acid synthesis reagent.
11. A single-stranded deoxyribonucleic acid primer that partially self-hybridizes to form
(1) a double-stranded target-non-specific region having a length of 4-21 nucleotides,
(2) a double-stranded target-specific region,
(3) a single-stranded target-specific region having a length of 4-20 nucleotides, and
(4) a hairpin loop region having a length of 4-30 nucleotides,
wherein the double-stranded target-non-specific region has a concentration-adjusted standard free energy that is within 10% of the concentration-adjusted standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
12. The single-stranded primer of claim 11 , wherein the single-stranded primer is comprised of deoxyribonucleic acid (DNA).
13. The single-stranded primer of claim 11 , wherein the single-stranded primer is comprised of ribonucleic acid (RNA).
14. A method of performing a multiplexed nucleic acid amplification reaction comprising amplifying multiple unique nucleic acid molecules using the primer of claim 11 .
15. A system comprising a nucleic acid target, a polymerase, and a single-stranded primer that partially self-hybridizes to form
(1) a double-stranded target-non-specific region,
(2) a double-stranded target-specific region,
(3) a single-stranded target-specific region, and
(4) a hairpin loop region, wherein the double-stranded target-non-specific region has a concentration-adjusted standard free energy that is within 10% of the concentration-adjusted standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
16. The system of claim 15 , wherein the double-stranded target non-specific region is about 4-21 nucleotide base pairs in length.
17. The system of claim 15 , wherein the single-stranded target-specific region is about 4-20 nucleotides in length.
18. The system of claim 15 , wherein the hairpin loop region is 4-30nucleotides in length.
19. The system of claim 15 , wherein the single-stranded primer is comprised of deoxyribonucleic acid (DNA).
20. The system of claim 15 , wherein the single-stranded primer is comprised of ribonucleic acid (RNA).
21. The system of claim 15 further comprising a nucleic acid target.