HELICASE DEPENDENT ISOTHERMAL AMPLIFICATION USING NICKING ENZYMES

• US 20130210019A1
• Filed: 08/16/2011
• Published: 08/15/2013
• 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
• Granted Patent: US 9,005,933 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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,

Wherein the method comprises amplifying said template nucleic acid using the helicase dependent amplification (HAD) 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 HAD reaction, wherein the HAD reaction is a thermophilic HAD (tHDA), wherein the sequence recognized by a nicking endonuclease is introduced into the template nucleic acid using an oligonucleotide primer comprising the sequence recognized by a nicking endonuclease and wherein the amplified sequence or sequences are shorter than 400 bp.

2. The method according to claim 1, wherein said primer comprising the sequence recognized by a nicking endonuclease comprises a 5′

tag sequence that does not hybridize to the template nucleic acid and the sequence recognized by a nicking endonuclease is in said tag sequence.

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

4. The method according to claim 1, wherein the helicase is selected from the group consisting of superfamily I helicases such as dda, perA, F-plasmid traI protein helicase and UvrD, superfamily II helicases such as recQ and NS3-helicase, superfamily III helicases such as AAV rep helicase, helicases from dnaB-like superfamily such as T7 phage helicase, and helicases form rho-like superfamily.

5. The method according to claim 1, wherein the polymerase 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 to claim 1, wherein the HAD amplification reaction is performed at a temperature above 50°

C., preferably between 50 and 70°

C., more preferably between 60 and 65°

C.

8. The method according to claim 1, additionally comprising an initial heat denaturation step.

9. The method according to claim 1, additionally comprising the step of detecting the amplification product.

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

13. Use of the method according to claim 1 for amplifying and optionally detecting a nucleic acid sequence.

11. A kit for amplifying a nucleic acid, comprising

a nicking endonuclease,

a helicase and

a DNA polymerase.

12. The kit of claim 11, further comprising

a buffering agent such as Tris, and/or

dNTPs, and/or

(d)ATP, and/or

A magnesium salt such as magnesium phosphate, and/or

NaCl, and/or

KCl.

14. Use of the kit according to claim 11 for amplifying and optionally detecting a nucleic acid sequence.