Patent ID: 8030000
Filing Date: 2011-10-04
Classification: B01L,C12N,C12Q,C12Y,G01N

Abstract:
1. An RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA, said process comprising the steps of: (a) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer; (b) contacting the first and second nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said first strand and form a 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 each other on the same template nucleic acid molecule; (c) extending the 3′ end of said first and second nucleoprotein primer with one or more polymerases and dNTPs to generate a first and second double stranded nucleic acid and a first and second displaced strand of nucleic acid; (d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached wherein the generated double stranded nucleic acid can serve as the double stranded target sequence wherein the RPA process is performed in the presence of at least one recombinase loading protein and at least one single stranded DNA binding protein, wherein said recombinase agent is a uvsX recombinase, said one or more polymerases comprises a DNA polymerase I with strand-displacing properties and which lacks 5′ to 3′ exonuclease activity, said at least one single stranded DNA binding protein is a gp32 single stranded DNA binding protein, and said at least one recombinase loading protein is a uvsY recombinase loading protein; and wherein the RPA process is performed in the presence of a crowding agent.