Recombinase Polymerase Amplification
First Claim
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 comprises 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 strandsuch 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%;
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 reaction is performed at a temperature of between 20°
C. and 50°
C.
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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 disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate 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.
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Citations
240 Claims
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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 comprises 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%; 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 reaction is performed at a temperature of between 20°
C. and 50°
C. - View Dependent Claims (53)
- end;
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2-52. -52. (canceled)
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54-222. -222. (canceled)
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223. A process comprising:
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providing an aqueous solution comprising (1) at least one recombinase; (2) at least one single stranded DNA binding protein; (3) at least one DNA polymerase; (4) dNTPs or a mixture of dNTPs and ddNTPs; (5) a reducing agent; (6) ATP or ATP analog; (7) at least one recombinase loading protein; and (8) a crowding agent; and freeze-drying the aqueous solution. - View Dependent Claims (224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240)
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Specification