Monitoring recombinase polymerase amplification mixtures
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.
1. A process comprising:
(a) providing a recombinase polymerase amplification reaction mixture comprising
a recombinase,
a single-stranded DNA binding protein (SSB), and
one or more oligonucleotides;
(b) contacting in a solution the recombinase polymerase amplification reaction mixture with a template nucleic acid;
(c) maintaining the reaction mixture under conditions that allow for the production of nucleic acid amplification products in the reaction mixture; and
(d) detecting particles associated with the nucleic acid amplification products in the reaction mixture.
2. The process of claim 1 , wherein the detecting is performed within 10 minutes of when the maintaining begins.
3. The process of claim 1 , wherein the reaction mixture comprises a crowding agent.
4. The process of claim 1 , wherein the detecting comprises determining a number or proportion of particles associated with nucleic acid amplification products.
5. The process of claim 1 , wherein the detecting comprises detecting single particles associated with two or more distinct nucleic acid amplification products.
6. The process of claim 1 , wherein the particles are about 0.5-20 μm in size.
7. The process of claim 1 , wherein the particles are detected using fluorescence from the particles.
8. The process of claim 1 , wherein the particles are detected without using fluorescence from the particles.
9. 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.
10. The process of claim 3 , wherein the crowding agent comprises polyethylene glycol, polyvinyl alcohol, dextran and/or Ficoll.
11. The process of claim 3 , wherein the crowding agent comprises polyethylene glycol.
12. The process of claim 11 , wherein the polyethylene glycol is selected from the group consisting of PEG1450, PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, PEG40000 and combinations thereof.
13. The process of claim 3 , wherein the crowding agent is present in the mixture at a concentration between 1 to 12% by weight or by volume of the mixture.
14. The process of claim 1 , wherein the recombinase comprises a RecA or UvsX protein.
15. The process of claim 1 , wherein the single-stranded DNA binding protein (SSB) comprises a prokaryotic SSB protein or a SSB protein derived from a myoviridae phage.
16. The process of claim 1 , wherein at least one of the one or more oligonucleotides comprises a detectable label.
17. The process of claim 1 , wherein the particles comprise one or more of the recombinase, the single-strand binding protein (SSB), and at least one of the one or more oligonucleotides.
18. 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, and a reverse transcriptase.
19. The process of claim 1 , wherein the particles are about 1-10 μm in size.
20. The process of claim 1 , wherein detecting particles in the mixture comprises the use of microscopy.
21. The process of claim 1 , wherein detecting particles in the mixture comprises the use of flow cytometry.
22. The process of claim 1 , wherein the particles comprise one or more of the recombinase, the single-strand binding protein, at least one of the one or more oligonucleotides, and a template nucleic acid.
23. The process of claim 1 , wherein the detecting comprises determining a number or proportion of particles which are not associated with nucleic acid amplification products.
24. The process of claim 20 , further comprising the use of a camera.
25. The process of claim 21 , further comprising the use of a camera.
26. The process of claim 1 , wherein detecting particles comprises the use of a camera.
27. The process of claim 3 , wherein the crowding agent is present at a concentration of between 2% and 8%.
28. The process of claim 27 , wherein the crowding agent is present at a concentration of between 4% and 5.5%.
29. The method of claim 15 , wherein the single-stranded DNA binding protein (SSB) comprises a T4 gp32 protein or a Rb69 gp32 protein.
30. The method of claim 15 , wherein the single-stranded DNA binding protein (SSB) comprises an E. coli SSB.
31. The process of claim 11 , wherein the polyethylene glycol is a PEG compound with molecular weight between 15,000 and 20,000 daltons.