Chimeric primers with hairpin conformations and methods of using same

1. A nucleic acid molecule comprising:

(a) a target-specific primer sequence;

(b) an anti-tag sequence 5′ of the target-specific primer sequence;

(c) a tag sequence 5′ of the anti-tag sequence; and

(d) a blocker between the anti-tag sequence and the tag sequence.

2. The nucleic acid molecule of claim 1 , wherein the anti-tag sequence and the tag sequence are each from 8 to 30 nucleotides in length.

3. The nucleic acid molecule of claim 2 , wherein the anti-tag sequence is 12 nucleotides in length.

4. The nucleic acid molecule of claim 2 , wherein the anti-tag sequence and the tag sequence are each 24 nucleotides in length.

5. The nucleic acid molecule of claim 1 , wherein the blocker is an iSp18 moiety or an iMe-isodC moiety.

6. A composition comprising:

(a) a first nucleic acid molecule, wherein the first nucleic acid molecule is a first member of a primer pair, comprising:

(i) a first target-specific primer sequence;

(ii) an anti-tag sequence 5′ of the target-specific primer sequence;

(iii) a tag sequence 5′ of the anti-tag sequence; and

(iv) a blocker between the anti-tag sequence and the tag sequence;

(b) a second nucleic acid molecule, wherein the second nucleic acid molecule is a second member of a primer pair, comprising:

(i) a second target-specific primer sequence;

(ii) a universal anti-tag sequence 5′ of the target-specific primer sequence;

(iii) a universal tag sequence 5′ of the anti-tag sequence; and

(iv) a blocker between the anti-tag sequence and the tag sequence; and

(c) a third nucleic acid molecule comprising:

(i) a universal anti-tag sequence complementary to the universal tag sequence; and

(ii) a label.

7. The composition of claim 6 , wherein the label is a fluorescent label.

8. The composition of claim 6 , comprising a plurality of different primer pairs for the amplification of a plurality of different target sequences, and a plurality of different anti-tag nucleic acid molecules covalently attached to a plurality of encoded microspheres, wherein the plurality of different anti-tag molecules comprise anti-tag sequences that are complementary to the different tag sequences in the plurality of different primer pairs, and wherein the identity of each of the different anti-tag nucleic acid molecules can be determined from the encoding of the encoded microsphere to which it is covalently attached.

9. The composition of claim 6 , further comprising an anti-tag sequence covalently attached to a microsphere.

10. A composition comprising:

(a) a first primer pair comprising:

(i) a first primer comprising:

a first target-specific primer sequence;

a universal anti-tag sequence 5′ of the target-specific primer sequence;

a universal tag sequence 5′ of the universal anti-tag sequence;

a blocker between the universal anti-tag sequence and the universal tag sequence; and

a chromophore attached to the universal tag sequence; and

(ii) a second primer comprising:

a second target-specific primer sequence;

a universal anti-tag sequence 5′ of the target-specific primer sequence;

a universal tag sequence 5′ of the universal anti-tag sequence;

a blocker between the universal anti-tag sequence and the universal tag sequence; and

a chromophore attached to the universal tag sequence; and

(b) a labeled nucleic acid molecule comprising:

(i) a universal anti-tag sequence complementary to the universal tag sequences of the first primer pair; and

(ii) a chromophore capable of Forster Resonance Energy Transfer with the chromophores of the first primer pair.

11. The composition of claim 10 , wherein the tag and anti-tag sequences are each from 8 to 30 nucleotides in length.

12. The composition of claim 10 , wherein the blocker is an iSp18 moiety or an iMe-isodC moiety.