Recombinase polymerase amplification
View Patent ↗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) a T6 UvsX H66S protein,
(b) a gp32 protein, and
(c) a polymerase,
wherein the gp32 protein is derived from a myoviridae phage.
2. The composition of claim 1 , further comprising 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 , in which 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 1 , wherein the gp32 protein is Rb69 gp32.
7. The composition of claim 2 , wherein the UvsY and gp32 proteins are selected from the group consisting of:
(a) Rb69 UvsY and Rb69 gp32;
(b) Aeh1 UvsY and Rb69 gp32;
(c) T4 UvsY and Rb69 gp32; and
(d) Rb69 UvsY and T4 gp32.
8. The composition of claim 2 , wherein the gp32 and UvsY proteins are native, hybrid or mutant proteins from the same or different myoviridae phage sources.
9. The composition of claim 8 , wherein the gp32 and UvsY proteins are hybrid proteins and comprise one or more amino acid residues from two different species of myoviridae phage.
10. The composition of claim 1 , wherein the T4 UvsX H66S protein further comprises the addition of one or more glutamic acid residues at the C-terminus; and/or the addition of one or more aspartic acid residues at the C-terminus.
11. The composition of claim 1 , further comprising a crowding agent.
12. The composition of claim 11 , wherein the crowding agent is selected from the group comprising: polyethylene glycol, polyethylene oxide, polystyrene, Ficoll, dextran, PVP, and albumin.
13. The composition of claim 11 , wherein the crowding agent has a molecular weight of less than 200,000 daltons.
14. The composition of claim 11 , wherein the crowding agent is present in an amount of about 0.5% to about 15% w/v.
15. The composition of claim 1 , 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.
16. The composition of claim 3 , wherein the nucleic acid primer is a blocked primer.
17. The composition of claim 16 , further comprising an endonuclease selected from the group consisting of E. coli exonuclease III and E. coli endonuclease IV.
18. The composition of claim 1 , further comprising about 1 mM to about 8 mM divalent manganese ions and/or heparin.
19. The composition of claim 2 , wherein at least one of the UvsX, gp32, or UvsY proteins comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:123, and SEQ ID NO:124.
20. The composition of claim 19 wherein the UvsX protein comprises the amino acid sequence SEQ ID NO:105, wherein UvsY is a Rb69 UvsY, wherein the gp32 protein is a Rb69 gp32 and the polymerase is a DNA polymerase I.
21. A composition comprising:
(a) a T6 UvsX H66S protein,
(b) a gp32 protein,
(c) a polymerase,
(d) at least one nucleic acid primer, and
(e) a target nucleic acid,
wherein the gp32 protein is derived from a myoviridae phage.