ISOTHERMAL NUCLEIC ACID AMPLIFICATION

• US 20110123991A1
• Filed: 06/11/2009
• Published: 05/26/2011
• Est. Priority Date: 06/11/2008
• Status: Active Grant

• Current Assignee: Geneform Technologies Limited
• Original Assignee: Geneform Technologies Limited
• Inventors: Hoser, Mark Jay
• Granted Patent: US 9,062,344 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

An isothermal process for amplifying a nucleic acid target molecule that relies on an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide, wherein 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, wherein the oligonucleotide is a substrate for the strand invasion system.

Claims

1. An isothermal process for amplifying a nucleic acid target molecule that relies on an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide, wherein 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, wherein the oligonucleotide is a substrate for the strand invasion system.

2. An isothermal process for amplifying a double-stranded nucleic acid target molecule in accordance with claim 1 comprising the following steps:

(a) providing an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide, wherein 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, wherein the oligonucleotide is a substrate for the strand invasion system;

(b) applying the oligonucleotide to the target molecule and allowing it to invade the duplex thereby rendering some or all of the target molecule single-stranded;

(c) applying the upstream primer to the single-stranded region of the target molecule 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 primer to the single-stranded target molecule and extending the 3′

end of the downstream primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule;

(e) continuing the reaction through repetition of (b) to (d).

3. The method of claim 1 wherein at least a portion of the oligonucleotide is complementary to a portion of the target sequence intervening the upstream and downstream primers.

4. The method of claim 1 wherein the oligonucleotide comprises a single-stranded DNA molecule of at least 30 nucleotides.

5. The method of claim 1 wherein the strand invasion system comprises a recombinase system.

6. An isothermal process for amplifying a double-stranded nucleic acid target molecule in accordance with claim 2 comprising the following steps:

(a) providing;

(i) upstream and downstream primers, each comprising a single-stranded DNA molecule of less than 30 nucleotides, at least a portion of which is complementary to sequence of the target molecule;

(ii) an oligonucleotide comprising a single-stranded DNA molecule of at least 30 nucleotides, at least a portion of which is complementary to sequence of the target molecule intervening the forward and reverse primers;

(b) contacting the oligonucleotide (ii) with recombinase to enable it to invade the complementary region of the target molecule thereby rendering the complementary region of the target molecule and adjacent regions single-stranded;

(c) applying the upstream primer to the single-stranded region of the target molecule 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 primer to the single-stranded target molecule and extending the 3′

end of the downstream primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule;

(e) continuing the reaction through repetition of (b) to (d).

7. The method of claim 1 wherein the oligonucleotide has a non-extendable 3′

terminus.

8. The method of claim 5 wherein the recombinase system comprises the T4 gp32, UvsX and UvsY.

9. The method of claim 1 wherein the substrate for the strand invasion system facilitates the separation of the target template duplex or the product of primer extension onto the target nucleic acid.

10. The method of claim 9 wherein one or more additional oligonucleotides facilitate the separation of the target duplex by the intervening oligonucleotide.

11. The method of claim 10 wherein the additional oligonucleotide binds to the strand released by the oligonucleotide and branch migrates into the proximal duplex nucleic acid.

12. The method of claim 9 wherein the additional oligonucleotide is a downstream extension of the oligonucleotide, wherein the downstream extension is not competent substrate for a polymerase.

13. The method of claim 1 wherein the oligonucleotide comprises a downstream element at its 3′

terminus which is complementary to the target sequence and which is not a polymerase substrate.

14. The method of claim 1 which employs a strand displacing polymerase.

15. The method of claim 1 wherein the forward primer comprises sequence which overlaps with the oligonucleotide.

16. The method of claim 1 wherein the reverse primer comprises sequence which is complementary to sequence of the downstream element.

17. The method of claim 1 which further comprises monitoring the amplification by measuring a detectable signal.

18. An isothermal process for amplifying a double-stranded nucleic acid target molecule in accordance with claim 6 comprising the following steps:

(a) providing;

(i) upstream and downstream primers, each comprising a single-stranded DNA molecule of less than 30 nucleotides, at least a portion of which is complementary to sequence of the target molecule;

(ii) an oligonucleotide comprising a single-stranded DNA molecule of at least 30 nucleotides, at least a portion of which is complementary to sequence of the target molecule intervening the forward and reverse primers, and further comprising a downstream element at its 3′

terminus which is complementary to sequence of the target molecule and which is not a polymerase substrate;

(b) contacting the oligonucleotide (ii) with recombinase to enable it to invade the complementary region of the target molecule thereby rendering the complementary region of the target molecule and adjacent regions single-stranded;

(c) applying the upstream primer to the single-stranded region of the target molecule 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 primer to the single-stranded target molecule and extending the 3′

end of the downstream primer with polymerase and dNTPs to produce a further double-stranded nucleic acid target molecule;

(e) continuing the reaction through repetition of (b) to (d).

19. A kit for isothermally amplifying a nucleic acid target molecule comprising an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide, wherein 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, wherein the oligonucleotide is a substrate for the strand invasion system.