Patent ID: 7435561

Claim:
A RPA process of DNA amplification of a target nucleic acid molecule comprising a first and a second strand of DNA, comprising the steps of: (a) contacting a recombinase agent with a first and a second nucleic acid primer and a third extension blocked primer which extension blocked primer comprises one or more noncomplementary or modified internal residue to form a first, second and third nucleoprotein primer; (b) contacting the first and second nucleoprotein primers to said double stranded target nucleic acid thereby forming a first double stranded structure between said first nucleoprotein primer and said first strand of DNA at a first portion of said first strand and a second double stranded structure between said second nucleoprotein primer and said second strand of DNA 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 each other on the same target nucleic acid molecule with a third portion of target nucleic acid between said 3′ ends; (c) extending the 3′ end of said first nucleoprotein primer and second nucleoprotein primer with one or more polymerases and dNTPs to generate a first amplified target nucleic acid with an internal region comprising the third portion of nucleic acid; (d) contacting said amplified target nucleic acid to said third nucleoprotein primer to form a third double stranded structure at the third portion of said amplified target nucleic acid in the presence of a nuclease; wherein said nuclease specifically cleaves said noncomplementary or modified internal residue only after the formation of said third double stranded structure to form a third 5′ primer double stranded structure and a third 3′ extension blocked primer double stranded structure; (e) extending the 3′ end of said third 5′ primer with one or more polymerase and dNTP to generate a second double stranded amplified nucleic acid which comprises said first nucleic acid primer and said third 5′ primer; (f) continuing the reaction through repetition of (b) through (e) until a desired degree of the second double stranded amplified nucleic acid is reached.