Recombinase polymerase amplification
View Patent ↗This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.
1. A composition comprising;
at least one recombinase agent;
at least one single stranded DNA binding protein;
at least one reverse transcriptase;
at least one DNA polymerase; and
a crowding agent.
2. The composition of claim 1 , wherein the at least one recombinase is selected from the group consisting of UvsX, RecA, and combinations thereof.
3. The composition of claim 1 , wherein the at least one single stranded DNA binding protein is selected from the group consisting of E. coli SSB, T4 gp32 and combinations thereof.
4. The composition of claim 1 , wherein the at least one polymerase comprises a DNA polymerase with strand displacing properties.
5. The composition of claim 1 , wherein the DNA polymerase is selected from the group consisting of E. coli DNA polymerase I Klenow fragment, B. stearothermophilus polymerase (Bst), B. subtilis Phi-29 polymerase, and B. subtilis polymerase I (Bsu).
6. The composition of claim 1 , wherein the crowding agent is present at a concentration between 1 to 12% of a weight or a volume of the composition.
7. The composition of claim 1 , wherein the crowding agent is selected from the group consisting of polyethylene glycol (PEG), dextran and Ficoll.
8. The composition of claim 7 , wherein the PEG is PEG1450, PEG3000, PEG8000 or PEG10000.
9. The composition of claim 7 , wherein the PEG has a molecular weight between 15000 and 20000.
10. The composition of claim 1 , further comprising ATP or an ATP analog, dNTP(s), or T4 bacteriophage UvsY.
11. The composition of claim 10 , wherein the dNTP(s) is/are selected from the group consisting of dATP, dGTP, dCTP and dTTP.
12. The composition of claim 10 , wherein the ATP or ATP analog is selected from the group consisting of ATP, ATP-γ-S, ATB-β-S, ddATP, and a combination thereof.
13. The composition of claim 1 , the composition further comprising a first nucleic acid primer and optionally a second nucleic acid primer.
14. The composition of claim 13 , wherein the first nucleic acid primer, the second nucleic acid primer, or both is labeled with a detectable label selected from the group consisting of enzymes, enzyme substrates, coenzymes, enzyme inhibitors, fluorescent markers, chromophores, luminescent markers, radioisotopes, and a member of a binding pair.
15. The composition of claim 1 , the composition further comprising a nuclease.
16. The composition of claim 15 , wherein the nuclease is endonuclease IV (Nfo) or 8-oxoguanine DNA glycosylase (fpg).
17. The composition of claim 1 , further comprising a buffer.