Methods and systems for nucleic acid amplification

1. A method for amplifying a nucleic acid sample, comprising:

a) providing a support comprising a first primer;

b) providing a first double-stranded nucleic acid molecule comprising a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is at least partially complementary to said first single-stranded nucleic acid molecule, wherein the first double-stranded nucleic acid molecule is derived from the nucleic acid sample;

c) at least partially denaturing the first double-stranded nucleic acid molecule by binding a first invader species free from said support to at least a portion of the first single-stranded nucleic acid molecule or at least a portion of the second single-stranded nucleic acid molecule, which binding exposes a first segment of the first single-stranded nucleic acid molecule that is complementary to the first primer; and

d) coupling the first primer to the first segment of the first single-stranded nucleic acid molecule and performing a first primer extension reaction using the first primer to generate a second double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and a third single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule, wherein the first primer extension reaction separates the second single-stranded nucleic acid molecule from the first single-stranded nucleic acid molecule.

2. The method of claim 1 , further comprising:

e) at least partially denaturing the second double-stranded nucleic acid molecule by binding a second invader species free from said support to at least a portion of the first single-stranded nucleic acid molecule or at least a portion of the third single-stranded nucleic acid molecule, which binding exposes a second segment of the first single-stranded nucleic acid molecule that is complementary to a second primer.

3. The method of claim 2 , further comprising:

f) providing the second primer; and

g) coupling the second primer to the second segment of the first single-stranded nucleic acid molecule and performing a second primer extension reaction using the second primer to generate a third double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and a fourth single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule, wherein the second primer extension reaction separates the third single-stranded nucleic acid molecule from the first single-stranded nucleic acid molecule.

4. The method of claim 3 , further comprising repeating f) and g) to generate one or more additional double-stranded nucleic acid molecules coupled to the support.

5. The method of claim 1 , wherein b) and c) are completed in solution.

6. The method of claim 2 , wherein the first primer has the same nucleic acid sequence as the second primer.

7. The method of claim 2 , wherein the first primer and the second primer have different nucleic acid sequences.

8. The method of claim 1 , wherein the first invader species is selected from the group consisting of an oligonucleotide, a nucleic acid comprising a locked nucleic acid (LNA), a nucleic acid comprising a peptide nucleic acid (PNA), and a sequence-specific single-stranded nucleic acid binding protein.

9. The method of claim 8 , wherein the first invader species is an oligonucleotide that cannot be extended in a primer extension reaction.

10. The method of claim 1 , wherein the support is a bead, an array pixel, a sensor, a particle, or an electrode.

11. The method of claim 1 , wherein the second single-stranded nucleic acid molecule is blocked such that it is not a template in a primer extension reaction.

12. The method of claim 1 , wherein the first double-stranded nucleic acid molecule is provided by subjecting a parent double-stranded nucleic acid molecule that is derived from the nucleic acid sample to one or more cycles of a nucleic acid amplification reaction, and wherein the first double-stranded nucleic acid molecule is an amplicon of the parent double-stranded nucleic acid molecule generated during the one or more cycles of the nucleic acid amplification reaction.

13. The method of claim 12 , wherein the one or more cycles of the nucleic acid amplification reaction are completed with the aid of a second invader species.

14. The method of claim 13 , wherein at least one of the first invader species and the second invader species is an oligonucleotide that cannot be extended in a primer extension reaction.

15. The method of claim 13 , wherein the one or more cycles of the nucleic acid amplification reaction are completed with the use of a solution-phase primer that is of the same nucleic acid sequence of the first primer.

16. The method of claim 1 , wherein the first primer extension reaction is completed with the aid of a strand-displacing polymerase.

17. The method of claim 1 , wherein the first invader species is an oligonucleotide that is bound to at least a portion of the first single-stranded nucleic acid molecule or at least a portion of the second single-stranded nucleic acid molecule with the aid of a recombinase.

18. The method of claim 1 , wherein the first primer cannot function as an invader species.

19. The method of claim 1 , wherein the nucleic acid sample is amplified isothermally.

20. The method of claim 1 , wherein the nucleic acid sample is clonally amplified.