COMPOSITIONS OF TOEHOLD PRIMER DUPLEXES AND METHODS OF USE

1 . A partially double-stranded primer comprised of first and second nucleic acid strands arranged into

(1) one double-stranded target-non-specific region,

(2) one double-stranded target-specific region, and

(3) one single-stranded target-specific region contributed to by the first nucleic acid strand,

wherein the double-stranded target-non-specific region has a standard free energy approximately equal to the standard free energy for the single-stranded target-specific region bound to a target nucleic acid.

2 . The partially double-stranded primer of claim 1 , wherein the second nucleic acid strand comprises a non-extendable nucleotide at its 3′ end and/or the first nucleic acid strand comprises a non-natural nucleotide at or near the 3′ end of its target-non-specific region.

3 . The partially double-stranded primer of claim 2 , wherein the non-extendable nucleotide is a non-natural nucleotide or a dideoxy nucleotide, optionally wherein the non-natural nucleotide is iso-C, iso-G or deoxyuridine.

4 . (canceled)

5 . The partially double-stranded primer of claim 1 , wherein the double-stranded target non-specific region is about 4-21 nucleotides in length and/or the single-stranded target-specific region is about 4-20 nucleotides in length.

6 . (canceled)

7 . The partially double-stranded primer of claim 1 , wherein the first and second nucleic acid strands are comprised of DNA or RNA.

8 . A system comprising

a nucleic acid target,

a polymerase, and

a partially double-stranded primer comprising a first and a second nucleic acid strand arranged into

(1) one double-stranded target-non-specific region,

(2) one double-stranded target-specific region, and

(3) one single-stranded target-specific region contributed to by the first nucleic acid strand.

9 . The system of claim 8 , wherein the nucleic acid target is a single-stranded.

10 . The system of claim 8 , wherein the nucleic acid target is DNA or RNA.

11 . The system of claim 8 , wherein the system comprises a plurality of different partially double-stranded primers.

12 . The system of claim 8 , wherein the system comprises at least two partially double-stranded primers which together can be used to amplify a region of the nucleic acid target.

13 . The system of claim 8 , wherein the nucleic acid target comprises repetitive sequence, secondary structure and/or high GC content.

14 . The system of claim 8 , wherein the nucleic acid target is present in a plurality of different nucleic acid targets, optionally wherein the nucleic acid target is present as a single copy or in low copy in a plurality of different nucleic acid targets.

15 . (canceled)

16 . A method comprising

contacting a partially double-stranded primer of claim 1 to a sample, and detecting hybridization of the primer to a target in the sample.

17 . The method of claim 16 , wherein the partially double-stranded primer is labeled with a detectable moiety, optionally wherein the detectable moiety comprises a fluorophore or a radioisotope.

18 . (canceled)

19 . The method of claim 16 , wherein the target is present as a single copy in the sample.

20 . A method comprising

hybridizing a single-stranded target-specific region of a first strand of a partially double-stranded primer of claim 1 to a nucleic acid target, thereby dissociating the first strand of the primer from a second strand of the primer, and

extending the first strand at its 3′ end, in a target-complementary manner, in the presence of a polymerase.

21 . A method comprising

performing a nucleic acid synthesis reaction in the presence of a nucleic acid target, a polymerase, and one or more partially double-stranded primers of claim 1 .

22 . The method of claim 21 , wherein the nucleic acid synthesis reaction is a nucleic acid amplification reaction, optionally a polymerase chain reaction (PCR), or wherein the nucleic acid synthesis reaction is a transcription reaction, optionally a reverse transcription reaction.

23 .- 26 . (canceled)

27 . A kit comprising

one or more partially double-stranded primers of claim 1 , and

one or more nucleic acid synthesis reagents.

28 .- 29 . (canceled)

30 . A kit comprising

a first single-stranded nucleic acid in a first container, and

a second single-stranded nucleic acid that is complementary to a region of the first single-stranded nucleic acid, in a second container,

wherein, when the first and second single-stranded nucleic acids are hybridized to each other, a partially double-stranded nucleic acid is formed that comprises

(1) a double-stranded target-non-specific region,

(2) a double-stranded target-specific region, and

(3) a single-stranded target-specific region contributed to by the first nucleic acid,

wherein the first single-stranded nucleic acid comprises a non-natural nucleotide and/or the second single-stranded nucleic acid comprises a non-extendable nucleotide at its 3′ end.

31 .- 33 . (canceled)

34 . A single stranded primer that partially self-hybridizes to form

(1) one or more double-stranded target-non-specific region,

(2) one or more double-stranded target-specific region,

(3) one or more single-stranded target-specific region, and

(4) one or more hairpin loop regions,

wherein the one or more double-stranded target-non-specific region has a concentration-adjusted standard free energy approximately equal to the concentration-adjusted standard free energy for the one or more single-stranded target-specific region bound to a target nucleic acid.

35 . A method of performing a multiplexed nucleic acid amplification reaction comprising amplifying multiple unique nucleic acid molecules using a primer of claim 1 .