Patent ID: 8426134

Claim:
A recombinase polymerase amplification process of DNA amplification of a double stranded target nucleic acid molecule comprising a first strand of DNA and second strand of DNA, the process comprising: (a) contacting in a solution a uvsX recombinase agent with a first nucleic acid primer and a second nucleic acid primer to form a first nucleoprotein primer and a second nucleoprotein primer, wherein each of the first and second nucleic acid primers comprise a single stranded region at its 3′ end; (b) contacting the first and the second nucleoprotein primers with the double stranded target nucleic acid molecule thereby forming: (1) a first double-stranded structure at a first portion of the first strand and (2) a second double stranded structure at a second portion of the second strand such that the 3′ end of each of the first nucleoprotein primer and the second nucleoprotein primer are oriented toward one another on the same double-stranded template nucleic acid molecule; (c) extending the 3′ end of each of the first and second nucleoprotein primers with dNTPs and one or more DNA polymerases with strand-displacing properties to generate a first double-stranded nucleic acid product and a second double-stranded nucleic acid product and a first displaced strand of nucleic acid product and a second displaced strand of nucleic acid product; and (d) repeating (b) and (c) to amplify the double stranded target nucleic acid molecule; wherein the solution further comprises a gp32 single-stranded DNA binding protein; wherein the solution further comprises a recombinase loading factor uvsY at a concentration of between 0.2 and 8 micromolar; wherein the solution further comprises a crowding agent at a concentration of between 1% and 12%; wherein the one or more DNA polymerase lack 5′ to 3′ exonuclease activity and lack FLAP endonuclease activity; wherein the solution further comprises a hydrolysable nucleoside triphosphate; and wherein the process is performed at a temperature of between 20° C. and 50° C.