Isothermal nucleic acid amplification
The disclosure relates to an isothermal process for amplifying a double-stranded nucleic acid target molecule. The process comprises providing an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide. The upstream and downstream primers are not substrates for the strand invasion system during the amplification process and do not amplify the target molecule independently of the strand invasion system, and the oligonucleotide is a substrate for the strand invasion system.
1. An isothermal process for amplifying a double-stranded nucleic acid target molecule comprising the following steps:
(a) providing:
(i) a recombinase;
(ii) an upstream nucleic acid primer and a downstream nucleic acid primer that do not bind the double-stranded nucleic acid target molecule, are not substrates for the recombinase, and do not amplify the target molecule independently of the recombinase, wherein each of said upstream and downstream nucleic acid primers comprises a single-stranded DNA molecule of less than 25 nucleotides, wherein at least a portion of each nucleic acid primer is complementary to a sequence of the target molecule; and
(iii) an oligonucleotide comprising a single-stranded DNA molecule of at least 30 nucleotides, at least 24 of said nucleotides of said oligonucleotide are complementary to the target molecule in a region between upstream and downstream nucleic acid primer binding sites, and at least a portion of the oligonucleotide is substrate for the recombinase;
(b) contacting the oligonucleotide with the double-stranded target molecule in the presence of the recombinase and allowing said oligonucleotide to invade the target molecule so that the region of the target molecule complementary to the oligonucleotide and regions adjacent to a terminus of the complementary region are rendered single-stranded to form a first strand and a second strand;
(c) applying the upstream nucleic acid primer to the first strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the upstream nucleic acid primer with polymerase and dNTPs to produce a double-stranded nucleic acid target molecule;
(d) applying the downstream primer to the second strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the downstream nucleic acid primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule; and
(e) continuing the reaction through repetition of (b) to (d).
2. The process of claim 1 , wherein the oligonucleotide has a non-extendable 3′ terminus.
3. The process of claim 1 , wherein the recombinase comprises UvsX.
4. The process of claim 1 , wherein the oligonucleotide facilitates strand separation of the double-stranded nucleic acid target molecule or an amplification product from the target nucleic acid.
5. The process of claim 4 , wherein one or more additional oligonucleotides facilitate the separation of the target duplex by the oligonucleotide.
6. The process of claim 5 , wherein the additional oligonucleotide binds to the strand released by the oligonucleotide and branch migrates into the proximal duplex nucleic acid.
7. The process of claim 1 , wherein the oligonucleotide comprises a downstream element at its 3′ terminus which is complementary to the target molecule and which is not a polymerase substrate.
8. The process of claim 1 , which employs a strand displacing polymerase.
9. The process of claim 1 , wherein the upstream nucleic acid primer comprises a sequence which overlaps with the oligonucleotide.
10. The process of claim 7 , wherein the downstream nucleic acid primer comprises a sequence which is complementary to a sequence of the downstream element of the oligonucleotide.
11. The process of claim 1 , which further comprises monitoring the amplification by measuring a detectable signal.
12. An isothermal process for amplifying a double-stranded nucleic acid target molecule in accordance with claim 1 comprising the following steps:
(a) providing:
(i) a recombinase;
(ii) an upstream nucleic acid primer and a downstream nucleic acid primer that do not bind the double-stranded nucleic acid target molecule, are not substrates for the recombinase, and do not amplify the target molecule independently of the recombinase, wherein each of said upstream and downstream nucleic acid primers comprises a single-stranded DNA molecule of less than 25 nucleotides, wherein at least a portion of each nucleic acid primer is complementary to a sequence of the target molecule; and
(iii) an oligonucleotide comprising a single-stranded DNA molecule of at least 30 nucleotides, at least 24 of said nucleotides of said oligonucleotide are complementary to the target molecule in a region between upstream and downstream nucleic acid primer binding sites and at least a portion of the oligonucleotide is substrate for the recombinase, and further comprising a downstream element at its 3′ terminus which is complementary to a sequence of the target molecule and which is not a polymerase substrate;
(b) contacting the oligonucleotide with the double-stranded target molecule in the presence of the recombinase and allowing said oligonucleotide to invade the target molecule so that the region of the target molecule complementary to the oligonucleotide and regions adjacent to a terminus of the complementary region are rendered single-stranded to form a first strand and a second strand;
(c) applying the upstream nucleic acid primer to the first strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the upstream primer with polymerase and dNTPs to produce a double-stranded nucleic acid target molecule;
(d) applying the downstream nucleic acid primer to the second strand of the target molecule at a region adjacent to and within 20 nucleotides of the region complementary to the oligonucleotide and extending the 3′ end of the downstream primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule; and
(e) continuing the reaction through repetition of (b) to (d) for amplification of the target molecule.
13. The process of claim 3 , wherein UvsX is used with at least one recombinase accessory protein.
14. The process of claim 13 , wherein the at least one recombinase accessory protein is selected from the group consisting of gp32, UvsY, and a combination thereof.
15. The process of claim 14 , wherein the at least one recombinase accessory protein is gp32.
16. The process of claim 14 , wherein the recombinase accessory protein is UvsY.
17. The process claim of 9 , wherein 5 to 10 nucleotides of a terminus of the upstream nucleic acid primer overlap with the oligonucleotide.
18. The process claim of 1 , wherein 24 to 63 nucleotides of the oligonucleotide is complementary to the target molecule.
19. The process claim of 1 , wherein each of the upstream and downstream nucleic acid primers comprises a single-stranded DNA molecule of 12 to 25 nucleotides.
20. The process claim of 19 , wherein each of the upstream and downstream nucleic acid primers comprises a single-stranded DNA molecule of 15 to 23 nucleotides.