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 and a second primer;

b) coupling the first primer to a first single-stranded nucleic acid molecule that is derived from the nucleic acid sample;

c) performing a first primer extension reaction using the first primer to generate a first double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule;

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

e) coupling the second primer to the segment of the first single-stranded nucleic acid molecule and performing a second primer extension reaction using the second 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 second 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:

(f) coupling a third primer to the second single-stranded nucleic acid molecule and performing a third primer extension reaction using the third primer to generate a third double-stranded nucleic acid molecule comprising the second single-stranded nucleic acid molecule and a fourth single-stranded nucleic acid molecule that is complementary to at least a portion of the second single-stranded nucleic acid molecule.

3. The method of claim 2 , wherein in (d), the method further comprises binding the invader species to at least a portion of the first single-stranded nucleic acid molecule and binding another invader species to at least a portion of the second single-stranded nucleic acid molecule, wherein the binding of the another invader species exposes a segment of the second single-stranded nucleic acid that is complementary to the third primer, and wherein coupling the third primer to the second single-stranded nucleic acid molecule comprises coupling the third primer to the segment of the second single-stranded nucleic acid.

4. The method of claim 1 , wherein the invader species is an oligonucleotide that has a length of at least 30 nucleotides.

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

6. The method of claim 2 , wherein a number of molecules of the third primer is greater than a number of molecules of the first primer and a number of molecules of the second primer.

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

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

9. The method of claim 2 , wherein the third primer is in solution.

10. The method of claim 2 , wherein the support comprises the third primer.

11. The method of claim 1 , wherein the invader species is an oligonucleotide that cannot be extended via the incorporation of additional nucleotides.

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

13. The method of claim 1 , wherein the first single-stranded nucleic acid molecule, the second single-stranded nucleic acid molecule, or the third single-stranded nucleic acid molecule is not released from the support during any of a)-e).

14. 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 double-stranded nucleic acid molecule is derived from the nucleic acid sample, and wherein the second-single-stranded nucleic acid molecule comprises a nucleic acid sequence at least partially complementary to said first primer;

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 or second single-stranded nucleic acid molecule, which binding exposes a segment of the first single-stranded nucleic acid molecule that is complementary to a second primer separate from said first primer;

d) coupling the second primer to the segment of the first single-stranded nucleic acid molecule and performing a first primer extension reaction using the second 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; and

e) coupling the first primer to the separated second-single stranded molecule and performing a second primer extension reaction using the first primer to generate a third double-stranded nucleic acid molecule comprising the second single-stranded nucleic acid molecule and a fourth single-stranded nucleic acid molecule.

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

a) providing a support comprising a first primer;

b) coupling the first primer to a first single-stranded nucleic acid molecule that is derived from the nucleic acid sample;

c) performing a first primer extension reaction using the first primer to generate a first double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule;

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

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

16. The method of claim 15 , wherein, in d), the binding of the invader species to the at least a portion of the first or second single-stranded nucleic acid molecule exposes a segment of the first single-stranded nucleic acid molecule that is complementary to the second primer.

17. The method of claim 16 , wherein, in e), the method comprises coupling the second primer to the segment of the first single-stranded nucleic acid molecule and performing the second primer extension reaction using the second primer to generate the second double-stranded nucleic acid molecule comprising the first single-stranded nucleic acid molecule and the third single-stranded nucleic acid molecule that is complementary to at least a portion of the first single-stranded nucleic acid molecule.

18. The method of claim 17 , further comprising

f) coupling a third primer to the second single-stranded nucleic acid molecule and performing a third primer extension reaction using the third primer to generate a third double-stranded nucleic acid molecule comprising the second single-stranded nucleic acid molecule and a fourth single-stranded nucleic acid molecule that is complementary to at least a portion of the second single-stranded nucleic acid molecule.

19. The method of claim 18 , wherein the third primer is free from the support.

20. The method of claim 18 , wherein the third primer is coupled to the support.

21. The method of claim 15 , wherein, in d), the binding of the invader species to the at least a portion of the first or second single-stranded nucleic acid molecule exposes a segment of the second single-stranded nucleic acid molecule that is complementary to the second primer.

22. The method of claim 21 , wherein, in e), the method comprises coupling the second primer to the segment of the second single-stranded nucleic acid molecule and performing the second primer extension reaction using the second primer to generate the second double-stranded nucleic acid molecule comprising the second single-stranded nucleic acid molecule and the third single-stranded nucleic acid molecule that is complementary to at least a portion of the second single-stranded nucleic acid molecule.

23. The method of claim 22 , further comprising

f) coupling a third primer to the first single-stranded nucleic acid molecule and performing a third primer extension reaction using the third 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.

24. The method of claim 23 , wherein the third primer is free from the support.

25. The method of claim 23 , wherein the third primer is coupled to the support.

26. The method of claim 15 , wherein the second primer is provided free from the support.

27. The method of claim 15 , wherein the second primer is provided coupled to the support.

28. The method of claim 15 , wherein the support is a bead, an array pixel, a sensor, a particle, or an electrode.

29. The method of claim 15 , wherein the first primer extension reaction is completed with the aid of a strand-displacing polymerase.

30. The method of claim 15 , wherein the invader species is an oligonucleotide that is bound to at least a portion of the first or second single-stranded nucleic acid molecule with the aid of a recombinase.

31. The method of claim 15 , wherein the nucleic acid sample is amplified isothermally.

32. The method of claim 14 , further comprising:

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

33. The method of claim 32 , further comprising:

providing the third primer; and

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

34. The method of claim 14 , 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.

35. The method of claim 34 , wherein the first invader species is an oligonucleotide that cannot be extended by a primer extension reaction.

36. The method of claim 14 , wherein the support is a bead, an array pixel, a sensor, a particle, or an electrode.

37. The method of claim 14 , 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.

38. The method of claim 14 , wherein the first primer extension reaction is completed with the aid of a strand-displacing polymerase.

39. The method of claim 14 , wherein the first invader species is an oligonucleotide that is bound to at least a portion of the first or second single-stranded nucleic acid molecule with the aid of a recombinase.

40. The method of claim 14 , wherein the first primer and the second primer are not invader species.

41. The method of claim 14 , wherein the nucleic acid sample is amplified isothermally.