MONITORING RECOMBINASE POLYMERASE AMPLIFICATION MIXTURES

• US 20120258456A1
• Filed: 04/06/2012
• Published: 10/11/2012
• Est. Priority Date: 04/07/2011
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Inventors: Armes, Niall A., Piepenburg, Olaf, Greenwood, Catherine Jean
• Granted Patent: US 8,809,021 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A process includes providing a mixture that includes a recombinase, a single-strand binding protein, and one or more oligonucleotides; and detecting particles in the reaction mixture.

Claims

1. A process comprising:

(a) providing a mixture comprising a recombinase, a single-stranded DNA binding protein, and one or more oligonucleotides; and

(b) detecting particles in the mixture.

2. The process of claim 1, wherein the mixture comprises a crowding agent.

3. The process of claim 2, wherein the crowding agent comprises polyethylene glycol, polyvinyl alcohol, dextran and/or ficoll.

4. The process of claim 2, wherein the crowding agent comprises polyethylene glycol.

5. The process of claim 3, wherein the polyethylene glycol is selected from the group consisting of PEG1450, PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, PEG40000, PEG compound with molecular weight between 15,000 and 20,000 daltons, and combinations thereof

6. The process of claim 2, wherein the crowding agent is present in the mixture at a concentration between 1 to 12% by weight or by volume of the mixture.

7. The process of claim 1, wherein the recombinase comprises a RecA or UvsX protein.

8. The process of claim 1, wherein the single-stranded DNA binding protein comprises a prokaryotic SSB protein or a gp32 protein.

9. The process of claim 1, wherein at least one of the one or more oligonucleotides comprises a detectable label.

10. The process of claim 1, wherein the particles comprise one or more of the recombinase, the single-strand binding protein, and at least one of the one or more oligonucleotides.

11. The process of claim 1, wherein the mixture further comprises one or more of:

a DNA polymerase, a recombinase loading protein, dNTPs or a mixture of dNTPs and ddNTPs, a reducing agent, creatine kinase, a nuclease, a nucleic acid probe, a reverse transcriptase, and a template nucleic acid.

12. The process of claim 1, wherein the particles are about 1-10 μ

m in size.

13. The process of claim 1, wherein detecting particles in the mixture comprises the use of microscopy.

14. The process of claim 1, wherein detecting particles in the mixture comprises the use of a microfluidic device.

15. The process of claim 1, wherein detecting particles in the mixture comprises the use of flow cytometry.

16. The process of claim 1, wherein the particles are about 0.5-20 μ

m in size.

17. The process of claim 1, wherein the particles are detected using fluorescence from the particles.

18. The process of claim 1, wherein the particles are detected without using fluorescence from the particles.

19. The process of claim 1, wherein the particles comprise a first subset of particles and a second subset of particles, the first subset of particles is detected using fluorescence from the first subset of particles, and the second subset of particles are detected without using fluorescence from the second subset of particles.

20. A process comprising:

(a) providing a recombinase polymerase amplification reaction mixture;

(b) maintaining the reaction mixture under conditions that allow for the production of nucleic acid amplification products in the reaction mixture; and

(c) detecting particles associated with the nucleic acid amplification products in the reaction mixture.

21. The process of claim 20, wherein the detecting is performed within 10 minutes of when the maintaining begins.

22. The process of claim 20, wherein the reaction mixture comprises a crowding agent.

23. The process of claim 20, wherein the detecting comprises determining a number or proportion of particles associated with nucleic acid amplification products.

24. The process of claim 20, wherein the detecting comprises detecting single particles associated with two or more distinct nucleic acid amplification products.

25. The process of claim 20, wherein the particles are about 0.5-20 μ

m in size.

26. The process of claim 20, wherein the particles are detected using fluorescence from the particles.

27. The process of claim 20, wherein the particles are detected without using fluorescence from the particles.

28. The process of claim 20, wherein the particles comprise a first subset of particles and a second subset of particles, the first subset of particles is detected using fluorescence from the first subset of particles, and the second subset of particles are detected without using fluorescence from the second subset of particles.

29. A composition comprising

(a) a first population of particles comprising a first recombinase, a first single-strand binding protein, and a first oligonucleotide;

and

(b) a second population of particles comprising a second recombinase, a second single-strand binding protein, and a second oligonucleotide, wherein the first and second oligonucleotides are different.

30. The composition of claim 29, wherein at least one of the first and second oligonucleotides comprises a detectable label.

31. The composition of claim 29, wherein the first and second oligonucleotides comprise different detectable labels.

32. The composition of claim 29, wherein the particles are about 0.5-20 μ

m in size.

33. The composition of claim 29, wherein the particles can be detected using fluorescence from the particles.

34. The composition of claim 29, wherein the particles can be detected without using fluorescence from the particles.

35. The composition of claim 29, wherein the particles comprise a first subset of particles and a second subset of particles, the first subset of particles can be detected using fluorescence from the first subset of particles, and the second subset of particles can be detected without using fluorescence from the second subset of particles.