Helicase dependent isothermal amplification using nicking enzymes

• US 9,005,933 B2
• Filed: 08/16/2011
• Issued: 04/14/2015
• Est. Priority Date: 08/17/2010
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Korfhage, Christian, Grosshauser, Gerd, Rothmann, Thomas, Himmelreich, Ralf
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/817,174
• Publication Number:
  US 20130210019A1
• Time in Patent Office: 1,337 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase

Abstract

The present invention relates to a method for amplifying a template nucleic acid, wherein the method comprises amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction in the presence of a nicking endonuclease, and wherein said template nucleic acid comprises a sequence recognized by said nicking endonuclease or a sequence recognized by said nicking endonuclease is introduced into the template nucleic acid during the HDA reaction. The invention further pertains to a kit for amplifying a nucleic acid, comprising a nicking endonuclease, a helicase and a DNA polymerase.

Claims

1. A method for amplifying a template nucleic acid, comprising:

(a) providing a template nucleic acid,

(b) contacting the template nucleic acid with a helicase,

(c) hybridizing an oligonucleotide primer to the template nucleic acid, and

(d) amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction, said HDA reaction introducing a recognition sequence into the template nucleic acid,

said HDA reaction occurring in the presence of a nicking endonuclease that recognizes a nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the template nucleic acid at a specific location relative to the recognition sequence,

wherein the HDA reaction is a thermophilic HDA (tHDA),

and wherein the template nucleic acid is shorter than 400 bp.

2. The method according to claim 1, wherein said oligonucleotide primer comprises a 5′

tag sequence that does not hybridize to the template nucleic acid and the recognition sequence is in said tag sequence.

3. The method according to claim 1, wherein the template nucleic acid is a double-stranded template nucleic acid.

4. The method according to claim 1, wherein the helicase is selected from the group consisting of dda, pcrA, F-plasmid traI protein helicase, UvrD, recQ, NS3-helicase, AAV rep helicase, T7 phage helicase, and rho-like superfamily helicase.

5. The method according to claim 1, wherein a polymerase in the HDA is selected from the group consisting of Bst DNA polymerase, PyroPhage polymerase, DisplaceAce polymerase and Vent (exo-) Polymerase.

6. The method according to claim 1, wherein the nicking endonuclease is selected from the group consisting of Nb.BsmI, Nt.BstNBI, Nt.CviPII, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI and Nt.AlwI.

7. The method according claim 1, wherein the HDA amplification reaction is performed at a temperature above 50°

C.

8. The method according to claim 1, additionally comprising heating the template nucleic acid prior to the amplifying in (d).

9. The method according to claim 1, additionally comprising detecting a product of the amplifying.

10. The method according to claim 9, wherein the product is detected using gel electrophoresis, an intercalating dye, or a specific oligonucleotide probe.

11. A method for amplifying a template nucleic acid, comprising:

(a) providing a template nucleic acid,

(b) contacting the template nucleic acid with a helicase, and

(c) amplifying said template nucleic acid using the helicase dependent amplification (HDA) reaction, the template nucleic acid comprising a recognition sequence,

said HDA reaction occurring in the presence of a nicking endonuclease that recognizes a nucleotide sequence of a completely or partially double-stranded nucleic acid molecule and cleaves only one strand of the template nucleic acid at a specific location relative to the recognition sequence,

wherein the HDA reaction is a thermophilic HDA (tHDA), and

wherein the template nucleic acid is shorter than 400 bp.

12. The method according to claim 11, wherein the template nucleic acid is a double-stranded template nucleic acid.

13. The method according to claim 11, wherein the helicase is selected from the group consisting of dda, pcrA, F-plasmid traI protein helicase, UvrD, recQ, NS3-helicase, AAV rep helicase, T7 phage helicase, and rho-like superfamily helicase.

14. The method according to claim 11, wherein a polymerase in the HDA is selected from the group consisting of Bst DNA polymerase, PyroPhage polymerase, DisplaceAce polymerase and Vent (exo-) Polymerase.

15. The method according to claim 11, wherein the nicking endonuclease is selected from the group consisting of Nb.BsmI, Nt.BstNBI, Nt.CviPII, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI and Nt.AlwI.

16. The method according claim 11, wherein the HDA amplification reaction is performed at a temperature above 50°

C.

17. The method according to claim 11, additionally comprising heating the template nucleic acid prior to the amplifying in (c).

18. The method according to claim 11, additionally comprising detecting a product of the amplifying.

19. The method according to claim 18, wherein the product is detected using gel electrophoresis, an intercalating dye, or a specific oligonucleotide probe.