Compositions for recombinase polymerase amplification
View Patent ↗This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.
1. A composition comprising:
i. a polymerase;
ii. a temperature sensitive recombinase agent;
iii. at least one oligonucleotide primer comprising a modified 3′ end, wherein the modified 3′ end comprises a free 3′ hydroxyl moiety;
iv. a single strand binding protein; and
v. ATP, an ATP analogue, or another nucleoside triphosphate.
2. The composition of claim 1 , wherein the polymerase is derived from a prokaryotic, viral, phage or eukaryotic source.
3. The composition of claim 2 , wherein the polymerase is a prokaryotic polymerase.
4. The composition of claim 3 , wherein the polymerase is from E. coli.
5. The composition of claim 4 , wherein the polymerase is selected from the group consisting of E.coli polymerase I, E. coli polymerase II, E. coli polymerase III, E. coli polymerase IV and E. coli polymerase V.
6. The composition of claim 2 , wherein the polymerase is a eukaryotic polymerase.
7. The composition of claim 6 , wherein the eukaryotic polymerase is selected from the group consisting of pol-α, pol-β, pol-δ, and pol-ε.
8. The composition of claim 1 , wherein the recombinase agent is RecA, RadA, RadB or Rad51 or a functional analogue or homologue thereof.
9. The composition of claim 8 , wherein the recombinase agent is a homologue of RecA obtained from a thermophilic organism, a prokaryote, a vertebrate or a plant.
10. The composition of claim 9 , wherein the recombinase agent is a homologue of RecA obtained from Thermococcus koadkaraensis, Thermotoga matitima, Aquifex pyrophilus, Pyrococcus furiosus, Thermus aquaticus, Pyrobaculum islandicum or Thermus thermophilus, Salmonella typhimurium, Bacillus subtilis, Streptococcus pneumonia, Bacteroides fragilis, Proteus mirabilis Rhizobium meliloti, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Ustilago maydis, Homo sapiens, Xenopus laevis or broccoli.
11. The composition of claim 8 , wherein the recombinase agent is a peptide comprising residues 193-212 of E. coli RecA.
12. The composition of claim 1 , wherein the at least one oligonucleotide primer comprises DNA, RNA and/or PNA.
13. The composition of claim 1 , wherein the at least one oligonucleotide primer comprises a modified 3′ end, and optionally a modified 5′ end, and wherein the modification comprises a detectable label, a magnetic tag and/or a protein.
14. The composition of claim 13 , wherein the detectable label is selected from the group consisting of an enzyme, an enzyme substrate, a coenzyme, an enzyme inhibitor, a fluorescent marker, a quencher, a chromophore, a luminescent marker, a radioisotope and one member of a binding pair.
15. The composition of claim 1 , comprising a first and a second oligonucleotide primer wherein the first oligonucleotide primer, the second oligonucleotide primer, or both the first and the second oligonucleotide primers comprise a modified 3′ end, and optionally at least one modification selected from the group consisting of a modified base, a modified linker backbone, and a modified 5′ end.
16. The composition of claim 15 , wherein the first oligonucleotide primer is at least partially complementary to a first portion of a first strand of a double stranded target nucleic acid and the second oligonucleotide primer is at least partially complementary to a portion of a second strand of the double stranded target nucleic acid, and wherein the 3′ ends of the first and second oligonucleotide primers are pointed towards each other when hybridised to the target nucleic target.
17. The composition of claim 16 , further comprising one or more additional oligonucleotide primers designed to be internal to the first and second oligonucleotide primers.
18. The composition of claim 17 , wherein at least one of the additional oligonucleotide primers comprises at least one modification selected from the group consisting of a modified base, a modified linker backbone, a modified 5′ end, and a modified 3′ end.
19. The composition of claim 1 , comprising three oligonucleotide primers wherein any one, any two, or all three of the oligonucleotide primers comprises a modification selected from the group consisting of a modified base, a modified linker backbone, a modified 5′ end, and a modified 3′ end.
20. The composition of claim 1 , wherein the at least one oligonucleotide primer is partially single stranded and partially double stranded.
21. The composition of claim 20 , wherein the at least one oligonucleotide primer comprises a longer invading-strand which possesses a specific 3′ primer sequence and a shorter non-invading strand, which is complementary to the 5′ end of the longer invading strand.
22. The composition of claim 21 , wherein the invading strand, the non-invading strand, or both the invading strand and the non-invading strand are labeled with a detectable label.
23. The composition of claim 1 , wherein the ATP, an ATP analogue, or another nucleoside triphosphate is ATP and wherein the composition further comprises RecO, RecR and/or RecF.
24. The composition of claim 1 , wherein the ATP analogue is ATPγS, dATP, ddATP or UTP.
25. The composition of claim 24 , wherein the ATP analogue is ATPγS and wherein the composition further comprises a helicase or PolV.
26. The composition of claim 1 , further comprising one or more accessory agents selected from the group consisting of topoisomerase, resolvase, a sliding clamp, DNA Polymerase III holoenzyme complex, RuvA, RuvB, RuvC, RecG, PriA, PriB, DnaT, DnaC, DnaG and DNA ligase.
27. The composition of claim 1 , further comprising dATP, dTTP, dCTP, dGTP or derivatives and analogues thereof.
28. The composition of claim 1 , wherein the temperature sensitive recombinase agent has strand-invasion activity at a permissive temperature and no strand-invasion activity at a non permissive temperature.
29. The composition of claim 28 , wherein the strand invasion activity of the temperature-sensitive recombinase agent is terminated at a non-permissive temperature, and restored when the temperature is shifted from the non-permissive temperature to the permissive temperature.
30. A composition comprising:
i. a polymerase;
ii. a recombinase agent;
iii. at least one oligonucleotide primer comprising at least one modification selected from the group consisting of a modified base, a modified linker backbone, a modified 5′ end, or a modified 3′ end, wherein the at least one oligonucleotide primer is partially single stranded and partially double stranded;
iv. a single strand binding protein; and
v. ATP, an ATP analogue, or another nucleoside triphosphate.
31. The composition of claim 30 , wherein the at least one oligonucleotide primer comprises a longer invading-strand which possesses a specific 3′ primer sequence and a shorter non-invading strand, which is complementary to the 5′ end of the longer invading strand.
32. The composition of claim 31 , wherein the invading strand, the non-invading strand, or both the invading strand and the non-invading strand are labeled with a detectable label.
33. A composition comprising:
i. a polymerase
ii. a recombinase agent
iii. at least one oligonucleotide primer comprising a modified 3′ end, wherein the at least one oligonucleotide primer comprises a longer invading-strand which possesses a specific 3′ primer sequence and a shorter non-invading strand, which is complementary to the 5′ end of the longer invading strand
iv. a single strand binding protein; and
v. ATP, an ATP analogue, or another nucleoside triphosphate.