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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundres of megabases in length.
1. A process comprising:
(a) converting a single stranded DNA or RNA molecule into a double stranded nucleic acid molecule comprising a first and second strand of DNA;
(b) contacting a recombinase agent selected from the group consisting of uvsX and recA with a first and second nucleic acid primer to form a first and second nucleoprotein primer;
(c) contacting the first and the second nucleoprotein primer to said double stranded nucleic acid molecule thereby forming a first double-stranded structure at a first portion of said first strand and forming a second double stranded structure at a second portion of said second strand such that the 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward one another on the same double-stranded template nucleic acid molecule;
(d) extending the 3′ ends of said first and second nucleic acid primer with one or more polymerases and dNTPs to generate a first and second double-stranded nucleic acid and first and second displaced strands of nucleic acid; and
(e) continuing the reaction through repetition of (c) and (d) until a desired degree of amplification is reached, wherein said process at steps (c) and (d) is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.
2. The process of claim 1 wherein said first and second nucleic acid primers are selected from the group consisting of DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid, and combinations thereof.
3. The process of claim 1 wherein said process is performed in the presence of a single strand stabilizing agent selected from the group consisting of SSB protein, gp32 protein, derivatives thereof, and combinations thereof.
4. The process of claim 3 wherein said single stranded DNA stabilizing agent is present at a concentration of between 1 μM and 20 μM.
5. The process of claim 1 , wherein said crowding agent is selected from the group consisting of polyethylene glycols, polyvinyl alcohols, dextran, ficoll and combinations thereof.
6. The process of claim 5 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.
7. The process of claim 1 , wherein the crowding agent has a concentration of between 1 and 12% weight/volume of the reaction.
8. The process of claim 1 wherein said process is performed in the presence of a recombinase loading protein.
9. The process of claim 8 wherein the recombinase loading agent is at a concentration of between 0.2 μM and 8 μM.
10. The process of claim 1 wherein the one or more polymerases comprise at least one DNA polymerase which lacks 3′-5′ exonuclease activity.
11. The process of claim 1 wherein said one or more polymerases comprise a DNA polymerase with strand displacing properties.
12. The process of claim 1 , wherein said process is performed between 20° C. and 50° C.
13. The process of claim 1 wherein one or both nucleic acid primers is a single stranded primer.
14. The process of claim 13 , wherein said single stranded primer is labeled with a detectable moiety selected from the group consisting of a fluorochrome, an enzyme, a fluorescence quencher, an enzyme inhibitor, a radioactive label and combinations thereof.
15. The process of claim 3 , wherein said single strand stabilizing agent derivative is a hexahistidine (His), c-myc epitope, FLAG(R) octapeptide, Protein C, Tag-100, V5 epitope, Xpress or hemagglutinin (HA) tagged derivative.