Recombinase polymerase amplification
First Claim
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 sequencewherein 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.
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Abstract
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, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.
114 Citations
23 Claims
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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:
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(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; andwherein the RPA process is performed in the presence of a crowding agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23)
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16. 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:
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(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;wherein one or more of the primers is initially 3′
-blocked, and contains a tetrahydrofuranyl residue, and wherein an E. coli Nfo protein is present in said RPA process, wherein a blocked oligonucleotide forms duplex hybrids with a target DNA and is subsequently cleaved by the Nfo protein; and
wherein the polymerase extends a free 3′
end of a probe generated by Nfo cleavage. - View Dependent Claims (17, 18, 19)
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Specification