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 RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA,
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 at least one primer is partially single stranded and partially double stranded, wherein said primer is comprised of 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.
2. The process of claim 1 wherein said first nucleic acid primer comprise a first non invading strand and said second nucleic acid primer comprise a second non-invading strand and said first and second non-invading strand are complementary to each other.
3. The process of claim 2 wherein said invading strand, said non-invading strand, or both said invading strand and said non-invading strand are labeled with a detectable moiety.
4. The process of claim 3 wherein said detectable moiety is selected from the group consisting of a fluorochrome, an enzyme, a fluorescence quencher, an enzyme inhibitor, a radioactive label, and a combination thereof.
5. The process of claim 3 wherein both said invading strand and said non-invading strand are labeled and wherein the invading strand is labeled with a first fluorochrome or a first enzyme and wherein the non-invading strand is labeled with a second fluorochrome, a second enzyme, a fluorescence quencher or an enzyme inhibitor.
6. The process of claim 3 wherein both said invading strand and said non-invading strand are labeled and wherein the non-invading strand is labeled with a first fluorochrome or a first enzyme and wherein the invading strand is labeled with a second fluorochrome, a second enzyme, a fluorescence quencher or an enzyme inhibitor.
7. The process of claim 1 wherein step (d) causes the displacement of the non-invading strand.
8. The process of claim 2 wherein the invading strand and the non-invading strand are labeled and the separation of the invading strand and the non-invading strand is detected.
9. A process according to claim 1 in which the released strand of one partly double-stranded invading oligonucleotide is the complementary sequence to the released strand of the other double-stranded invading oligonucleotide.
10. A process according to claim 9 in which the released oligonucleotide is complementary to, and hybridizes to a single-stranded target template and prime DNA synthesis from its 3′ end.
11. A RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA,
comprising the steps of:
(a) contacting a recombinase agent with a first and a second single stranded 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 D loop structure at a first portion of said first strand and form a second D loop 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 recombinase agent is a temperature-sensitive recombinase agent with reversible strand invasion activity in which activity is terminated at a non-permissive temperature, and restored when the temperature is shifted from the non-permissive temperature to the permissive temperature, and wherein the non-permissive temperature of the temperature sensitive recombinase agent is 42° C. and the permissive temperature is 25° C.-37° C.