Patent ID: 7666598
Filing Date: 2010-02-23
Classification: C12Q

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