Recombinase polymerase amplification
View Patent ↗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.
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.
2. The process of claim 1 wherein said first or second nucleic acid primers have a length that is below the length required for optimal strand exchange to decrease nonspecific amplification reactions.
3. The process of claim 1 further comprising the step of ligating additional nucleic acid sequence to at least one end of said target nucleic acid before step (b) to reduce foldback priming or to modulate nucleoprotein recombination/elongation rate.
4. The process of claim 1 further comprising the step of monitoring total DNA concentration with a sensor during said process.
5. The process of claim 1 wherein said RPA process is started or controlled by a light-driven photodeprotection of one or more caged substance.
6. The process of claim 5 wherein said caged substance is caged ATP or caged oligonucleotides.
7. The process of claim 5 wherein the concentration of uncaged molecules are monitored.
8. The process of claim 1 wherein one or both of said primers comprise cytosine at greater than 75% of the bases of the primer.
9. The process of claim 1 which is performed in a gel matrix which restricts the mobility of the template nucleic acid and an RPA product.
10. The process of claim 9 wherein said gel matrix is a polony gel matrix.
11. A process of nested RPA comprising the steps of:
(a) performing an RPA reaction of claim 1 in the presence of a first pair of outer primers and a second pair of inner primers in said step (a);
wherein said first pair of outer primers comprise nucleic acid sequence such that the 3′ ends of said pair of outer primers are oriented toward each other on the same template nucleic acid molecule after step (b);
wherein said second pair of inner primers comprise nucleic acid sequence such that the 3′ ends of said pair of inner primers are oriented toward each other on the same template nucleic acid molecule after step (b),
further wherein said second pair of inner primers hybridize to the target nucleic acid at a location that is within the hybridization location of said first pair of outer primers.
12. The process of claim 11 wherein said process is performed in the presence of dUTP such that dUTP is incorporated into an RPA product wherein said RPA product is susceptible to a dUTP deglycosylase.
13. The process of claim 11 wherein said process is performed in the presence of an enzyme selected from the group consisting of DNA ligase, helicase, RNA polymerase, reverse transcriptase, FLAP endonuclease, 5′-3′ exonuclease, 3′-5′ exonuclease, restriction endonuclease, endonuclease and base-specific damaged base-specific nuclease.
14. The process of claim 11 wherein at least one said primer comprise additional nucleic acid sequences which permit optimal seeding and phasing properties of recombinase on the primer.
15. The process of claim 14 wherein the additional DNA sequences comprises more than 70% pyrimidines or more than 75% of cytosine residues.
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:
(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.
17. The process of claim 16 , wherein said blocked primer contains a tetrahydrofuranyl residue, a fluorophore, and a quenching minor groove binding dye, such that on forming duplex hybrids the quenching is decreased by virtue of a change in absorption properties of the minor groove binding dye such that a rise in fluorescence emission from the fluorophore increases as a consequence of DNA amplification.
18. The process of claim 16 , wherein step (a) is performed in the presence of a first pair of outer primers and a second pair of inner primers;
wherein said first pair of outer primers comprise nucleic acid sequence such that the 3′ ends of said pair of outer primers are oriented toward each other on the same template nucleic acid molecule after step (b);
wherein said second pair of inner primers comprise nucleic acid sequence such that the 3′ ends of said pair of inner primers are oriented toward each other on the same template nucleic acid molecule after step (b); and
further wherein said second pair of inner primers hybridize to the target nucleic acid at a location that is within the hybridization location of said first pair of outer primers
and wherein one or more of the outer or inner 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.
19. The process of claim 18 , wherein said blocked primer contains a tetrahydrofuranyl residue, a fluorophore, and a quenching minor groove binding dye, such that on forming duplex hybrids the quenching is decreased by virtue of a change in absorption properties of the minor groove binding dye such that a rise in fluorescence emission from the fluorophore increases as a consequence of DNA amplification.
20. The process of claim 1 , wherein the crowding agent is selected from the group consisting of polyethylene glycols, dextran, ficoll, and combinations thereof.
21. The process of claim 20 , wherein the crowding agent comprises polyethylene glycol.
22. The process of claim 1 , wherein the crowding agent is present at a concentration of between 1 and 12% by weight or by volume.
23. The process of claim 22 , wherein the crowding agent comprises polyethylene glycol.