Nucleic acid amplification

• US 10,329,544 B2
• Filed: 04/06/2016
• Issued: 06/25/2019
• Est. Priority Date: 05/13/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Li, Chieh-Yuan, Ruff, David, Chen, Shiaw-Min, O'Neil, Jennifer, Kasinskas, Rachel, Rothberg, Jonathan, Li, Bin, Lao, Kai Qin, Hinz, Wolfgang
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/091,717
• Publication Number:
  US 20160272954A1
• Time in Patent Office: 1,175 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

In some embodiments, the present teachings provide methods for nucleic acid amplification, comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, methods for nucleic acid amplification include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, methods for nucleic acid amplification include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the methods for nucleic acid amplification employ an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, methods for nucleic acid amplification can be conducted in a single reaction vessel. In some embodiments, methods for nucleic acid amplification can be conducted in a single continuous liquid phase of a reaction mixture, without need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, methods for nucleic acid amplification comprise a amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer can include a sieving agent and/or a diffusion-reducing agent.

Claims

1. A composition for nucleic acid amplification comprising a recombinase, a recombinase accessory protein, a plurality of supports having a plurality of forward primers attached thereto, a plurality of reverse primers in solution, and a T5 or T7 DNA polymerase, wherein the plurality of forward primers have identical nucleotide sequences, wherein the plurality of reverse primers have identical nucleotide sequences, wherein the composition is a single continuous liquid phase comprising a sieving agent, wherein the single continuous liquid phase is not within a gel or matrix, wherein the T5 or T7 DNA polymerase has reduced exonuclease activity compared to wild-type T5 or T7 polymerase, respectively, and wherein if the polymerase is T7 polymerase, the composition further comprises thioredoxin.

2. The composition of claim 1, wherein the composition comprises a T7 DNA polymerase having reduced 3′

-5′

exonuclease activity.

3. The composition of claim 2, wherein the T7 DNA polymerase has an E7A, a D5A, or both an E7A and a D5A mutation, wherein the numbering is relative to the amino acid sequence of SEQ ID NO:

1.

4. The composition of claim 3, wherein the T7 DNA polymerase is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, and SEQ ID NO;

4.

5. The composition of claim 1, wherein the composition further comprises a Bsu polymerase or a Sau polymerase.

6. The composition of claim 1, wherein the composition further comprises a single-stranded binding protein.

7. The composition of claim 6, wherein the single-stranded binding protein is gp32.

8. The composition of claim 1, wherein the recombinase is a UvsX protein and the recombinase accessory protein is a UvsY protein.

9. The composition of claim 1, wherein the recombinase is a UvsX protein, the recombinase accessory protein is UvsY, and the composition further comprises a Sau polymerase.

10. The composition of claim 1, wherein the plurality of supports comprises a plurality of beads distributed on a plurality of sites within an array wherein each site is independently linked to an ISFET sensor capable of detecting the presence of a nucleotide incorporation byproduct.

16. The composition of claim 1, wherein at least two of the plurality of solid supports each comprise a different substantially monoclonal population of nucleic acids.

17. The composition of claim 16, wherein at least 90% of the nucleic acids in each substantially monoclonal population share at least 99% sequence identity.

18. The composition of claim 16, wherein the composition further comprises two or more polynucleotide templates in solution in the continuous liquid phase.

19. The composition of claim 1, wherein the sieving agent comprises a cellulose or other polysaccharide polymer.

11. A kit for nucleic acid amplification, comprising:

one or more solid supports;

a plurality of forward primers attached to the one or more solid supports, wherein the plurality of forward primers have identical nucleotide sequences;

a plurality of reverse primers in solution, wherein the plurality of reverse primers have identical nucleotide sequences;

a recombinase;

a sieving agent comprising a cellulose or other polysaccharide polymer;

a recombinase accessory protein;

and a T5 or T7 DNA polymerase, wherein the T5 or T7 DNA polymerase has reduced exonuclease activity compared to wild-type T5 or T7 polymerase, respectively, and wherein if the polymerase is T7 polymerase, the kit further comprises thioredoxin.

12. The kit of claim 11, wherein the kit comprises a T7 DNA polymerase having a reduced 3′

-5′

exonuclease activity, and wherein the T7 DNA polymerase has an E7A, a D5A, or both an E7A and a D5A mutation, wherein the numbering is relative to the amino acid sequence of SEQ ID NO;

1.

13. The kit of claim 11, wherein the kit further comprises a Bsu polymerase or a Sau polymerase.

14. The kit of claim 11, wherein the kit further comprises a single-stranded binding protein.

15. The kit of claim 11, wherein the recombinase is a UvsX protein and the recombinase accessory protein is a UvsY protein.