US 7,666,598 B2 · Recombinase polymerase amplification

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 carryover 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.

Claims134

Dependent claims

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.

13 more elements

A process of detecting the presence or absence of a recombinase polymerase amplification amplified nucleic acids product in a sample, comprising the steps of:

(a) contacting in solution a T4 recombinase agent with a first and second nucleic acid primer to form a first and second nucleoprotein primer, wherein said nucleic acid primers comprise a single stranded region at its 3′ end, wherein said first nucleic acid primer is labeled with a first member of a first binding pair, and wherein said second nucleic acid primer is labeled with a first member of a second binding pair;
(b) contacting the first and the second nucleoprotein primers to a sample suspected to contain an amplified target nucleic acid amplified by a recombinase polymerase amplification (RPA) process to form a complex with a first and a second nucleoprotein primer/target nucleic acid structure at a first portion and a second portion of said amplified target nucleic acid, respectively;
(c) contacting said complex with a first mobile solid support coated with a second member of said first binding pair and a second mobile solid support coated with a second member of said second binding pair;
(d) determining if said first mobile solid support is co-localized with said second mobile solid support to determine the presence of said amplification amplified nucleic acid product;
wherein the recombinase loading factor T4 uvsY is included in the reaction at a concentration of between 0.2 and 8 micromolar;
wherein a crowding agent is included in the RPA process at a concentration between 1% and 12% such that the crowding agent stimulates amplification;
wherein a hydrolysable nucleoside triphosphate is employed to support recombinase activity such that uvsX-loaded DNA filaments are capable of efficient disassembly as well as assembly.

4 more elements

A recombinase polymerase amplification process of DNA amplification of a double stranded target molecule comprising the steps of:

(a) contacting in solution a recombinase agent selected from the group consisting of uvsX and recA with a first and a second nucleic acid primer to form a first and a second nucleoprotein primers;
(b) contacting the first and second nucleoprotein primers to said double stranded target nucleic acid molecule thereby forming a first double stranded structure at a first portion of said target nucleic acid molecule and a second double stranded structure at a second portion of said target nucleic acid molecule such that 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the template nucleic acid molecule;
(c) contacting said target nucleic acid molecule to primosome assembly proteins and a clamp loader/polymerase holoenzyme complex to form a replication fork at said first portion of the target nucleic acid molecule and a second replication fork at said second portion of the target nucleic acid molecule;
(d) contacting said target molecule to a DNA polymerase III core, a DNA polymerase I, a primase, a helicase, and a ligase to allow coupled leading and lagging strand DNA synthesis and migration of each replication fork towards each other;
and
(e) repeating (b), (c) and (d) until a desired degree of amplification is reached;
wherein a gp32 single-stranded DNA binding protein is included at a concentration at least sufficient to saturate the first and second nucleic acid primers in solution at step (a);
wherein the recombinase loading factor uvsY or the E. coli recO and recR gene products are included in the reaction at a concentration of between 0.2 and 8 micromolar;
wherein a crowding agent is included at a concentration of at least 1% to 12% such that the crowding agent stimulates amplification;
wherein a hydrolysable nucleoside triphosphate is employed to support recombinase activity such that recombinase-loaded DNA filaments are capable of efficient disassembly as well as assembly.

7 more elements

Dependencies are inferred from the claim text.

Recombinase polymerase amplification

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Recombinase polymerase amplification

Life of this patent

Abstract

Claims134