Recombinase polymerase amplification
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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundres of megabases in length.
1. A dried recombinase polymerase amplification (RPA) reagent composition comprising:
(1) at least one recombinase;
(2) at least one polymerase;
(3) trehalose; and
(4) a crowding agent.
2. The dried RPA reagent composition of claim 1 , wherein the dried RPA reagent mixture is provided as freeze-dried or lyophilized powder, pellet or bead.
3. The dried RPA reagent composition of claim 1 , wherein the at least one recombinase is selected from the group consisting of uvsX, recA and combinations thereof.
4. The dried RPA reagent composition of claim 1 , wherein the at least one polymerase is a DNA polymerase I.
5. The dried RPA reagent composition of claim 1 , wherein the at least one polymerase includes at least one DNA polymerase which lacks 3 ′-5 ′ exonuclease activity.
6. The dried RPA reagent composition of claim 1 , wherein the at least one polymerase comprises a DNA polymerase with strand displacing properties.
7. The dried RPA reagent composition of claim 1 , further comprising a first primer and optionally a second primer.
8. The dried RPA reagent composition of claim 1 , further comprising a single stranded DNA binding protein (SSB).
9. The dried RPA reagent composition of claim 8 , wherein the single stranded DNA binding protein is a E. coli single stranded DNA binding protein, a T4 gp32 or combinations thereof.
10. The dried RPA reagent composition of claim 1 , further comprising a dNTP or a mixture of dNTPs and ddNTPs.
11. The dried RPA reagent composition of claim 1 , further comprising a reducing agent.
12. The dried RPA reagent composition of claim 1 , further comprising at least one recombinase loading protein.
13. The dried RPA reagent composition of claim 12 , wherein the at least one recombinase loading protein is selected from the group consisting of T4 uvsY, E. coli recO, E. coli recR and combinations thereof.
14. The dried RPA reagent composition of claim 1 , further comprising ATP or a hydrolysable ATP analog.
15. The dried RPA reagent composition of claim 1 , wherein, when reconstituted, the reconstituted mixture comprises trehalose at a concentration of 20 mM to 200 mM.
16. The dried RPA reagent composition of claim 1 , wherein, when reconstituted, the reconstituted mixture comprises trehalose at a concentration of 40 mM to 80 mM.
17. The dried RPA reagent composition of claim 1 , wherein, when reconstituted, the reconstituted mixture comprises uvsX recombinase at concentration of between 0.2 μM to 12 μM.
18. The dried RPA reagent composition of claim 12 , wherein, when reconstituted, the reconstituted mixture comprises a recombinase loading protein at a concentration of between 0.2 μM to 8 μM.
19. The dried RPA reagent composition of claim 9 , wherein, when reconstituted, the reconstituted mixture comprises a gp32 single stranded DNA binding protein at a concentration between 1 to 30 μM.
20. The dried RPA reagent composition of claim 10 , wherein, when reconstituted, the reconstituted mixture comprises dNTPs or a mixture of dNTPs and ddNTPs at a concentration of between 1-300 μM.
21. The dried RPA reagent composition of claim 1 , wherein the crowding agent is selected from the group consisting of polyethylene glycol, dextran, ficoll, and combinations thereof.