Methods and systems for nucleic acid amplification

• US 10,533,218 B2
• Filed: 10/05/2017
• Issued: 01/14/2020
• 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 C
• Application Number:
  US15/726,193
• Publication Number:
  US 20180094307A1
• Time in Patent Office: 831 Days
• 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;

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.