Methods and systems for nucleic acid amplification

• US 9,822,401 B2
• Filed: 04/16/2015
• Issued: 11/21/2017
• Est. Priority Date: 04/18/2014
• Status: Active Grant

• Current Assignee: Sequencing Health Incorporated
• Original Assignee: GenapSys, Inc.
• Inventors: Oberstrass, Florian
• Primary Examiner(s): Thomas, David
• Application Number:
  US14/688,764
• Publication Number:
  US 20150344943A1
• Time in Patent Office: 950 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: Y02A 90/40 Monitoring or fighting invasive species

Abstract

The disclosure provides methods and systems for nucleic acid amplification including isothermal nucleic acid amplification.

Claims

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.

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.

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.