Recombinase polymerase amplification
The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.
1. A composition, comprising:
a T6 UvsX H66S protein; and
a crowding agent.
2. The composition of claim 1 , further comprising one or more additional components selected from the group consisting of a gp32 protein derived from a myoviridae phage, a polymerase, and a UvsY protein.
3. The composition of claim 1 , further comprising at least one nucleic acid primer.
4. The composition of claim 1 , further comprising a target nucleic acid.
5. The composition of claim 2 , wherein the UvsY protein is derived from a myoviridae phage, and the myoviridae phage from which the gp32 and UvsY proteins are derived is selected from the group consisting of: T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3, and phage LZ2.
6. The composition of claim 2 , wherein the gp32 protein is Rb69 gp32.
7. The composition of claim 1 , wherein the crowding agent is selected from the group comprising: polyethylene glycol, polyethylene oxide, polystyrene, Ficoll, dextran, PVP, and albumin.
8. The composition of claim 1 , wherein the crowding agent has a molecular weight of less than 200,000 daltons.
9. The composition of claim 1 , wherein the crowding agent is present in an amount of about 0.5% to about 15% w/v.
10. The composition of claim 2 , wherein the polymerase is a large fragment polymerase selected from the group consisting of E. coli Pol I, Bacillus subtilis Pol I, Staphylococcus aureus Pol I, and homologues thereof.
11. The composition of claim 3 , wherein the nucleic acid primer is a blocked primer.
12. The composition of claim 1 , further comprising an endonuclease selected from the group consisting of E. coli exonuclease III and E. coli endonuclease IV.
13. The composition of claim 1 , further comprising about 1 mM to about 8 mM divalent manganese ions and/or heparin.
14. A method, comprising:
contacting a nucleic acid molecule with the composition of claim 1 .
15. A method, comprising:
amplifying a nucleic acid molecule with the composition of claim 2 .