Helicase-dependent amplification of nucleic acids

• US 20070207495A1
• Filed: 04/27/2007
• Published: 09/06/2007
• Est. Priority Date: 09/20/2002
• Status: Active Grant

• Current Assignee: BioHelix Corp. (Quidel Corporation)
• Original Assignee: New England Biolabs Incorporated
• Inventors: Xu, Yan, Kong, Huimin, Vincent, Myriam
• Granted Patent: US 7,829,284 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase

Abstract

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a helicase preparation and a DNA polymerase such that the amplification can be performed isothermally.

Claims

1. A method for helicase-dependent amplification of a target nucleic acid, comprising:

(a) adding to the target nucleic acid, a helicase preparation, comprising;

a helicase and a single strand binding protein unless the helicase is a thermostable helicase in which case the single strand binding protein is not required;

one or more oligonucleotide primer pairs; and

one or more polymerases; and

(b) amplifying the target nucleic acid wherein the amplification is dependent on the helicase preparation.

2. A method according to claim 1, wherein amplification is isothermal.

3. A method according to claim 1, wherein the target nucleic acid is a DNA.

4. A method according to claim 1, wherein the target nucleic acid is an RNA where the RNA is reverse transcribed to form a DNA.

5. A method according to claim 1, wherein the target nucleic acid is a double-stranded nucleic acid, the double-stranded nucleic acid having been denatured enzymatically or by heat prior to step (a).

6. A method according to claim 1, wherein the target nucleic acid has a size in the range of about 50 bp to 100 kb.

7. A method of claim 1, wherein the one or more oligonucleotide primer pairs are one pair of oligonucleotide primers and wherein a first primer in the pair hybridizes to the 5′

-end and a second primer in the pair hybridizes to 3′

-end of the target nucleic acid to be selectively amplified.

9. A method according to claim 1, wherein the one or more oligonucleotide primer pairs have a length and a GC content so that the melting temperature of the oligonucleotide primers is about 10°

-30°

C. above the reaction temperature of hybridization during amplification.

10. A method according to claim 9, wherein the DNA polymerase is selected from a Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase (Sequenase) and Bst polymerase Large fragment.

11. A method according to claim 10, wherein the DNA polymerase lacks 5′

to 3′

exonuclease activity.

12. A method according to claim 11, wherein the DNA polymerase possesses strand displacement activity.

13. A method according to claim 1, wherein the helicase preparation comprises a single helicase.

14. A method according to claim 1, wherein the helicase preparation comprises a plurality of helicases.

15. A method according to claim 1, wherein the helicase preparation comprises a 3′

to 5′

helicase.

16. A method according to claim 1, wherein the helicase preparation comprises a 5′

to 3′

helicase.

17. A method according to claim 1, wherein the helicase preparation comprises a superfamily 1 helicase.

18. A method according to claim 1, wherein the helicase preparation comprises a superfamily 4 helicase.

19. A method according to claim 1, wherein the helicase preparation is selected from a superfamily 2 helicase, a superfamily 3 helicase, and an AAA⁺ helicase.

20. A method according to claim 1, wherein the helicase preparation comprises a hexameric helicase.

21. A method according to claim 1, wherein the helicase preparation comprises a monomeric or dimeric helicase.

22. A method according to claim 1, wherein the helicase preparation comprises a UvrD helicase or homolog thereof.

23. A method according to claim 22, wherein the UvrD helicase comprises a thermostable helicase or homolog thereof.

24. A method according to claim 1, wherein the helicase preparation comprises one or more helicases selected from the group consisting of:

a UVrD helicase, a recBCD helicase, E. coli UvrD helicase, Tte-UvrD helicase, T7 Gp4 helicase, RecBCD helicase, DnaB helicase, MCM helicase, Rep helicase, RecQ helicase, PcrA helicase, SV40 large T antigen helicase, Herpes virus helicase, yeast Sgs1 helicase, DEAH_ATP-dependent helicases and Papillomavirus helicase E1 protein and homologs thereof.

25. A method according to claim 22, wherein the UvrD helicase is E. coli UvrD helicase.

26. A method according to claim 23, wherein the thermostable helicase is Tte-UvrD helicase.

27. A method according to claim 1, wherein the helicase preparation comprises a RecBCD helicase.

28. A method according to claim 14, wherein the helicase preparation comprises T7 gene 4 helicase and E. coli UvrD helicase.

29. A method according to claim 1, wherein the energy source in the helicase preparation is selected from adenosine triphosphate (ATP), deoxythymidine triphosphate (dTTP) or deoxyadenosine triphosphate (dATP).

30. A method according to claim 29, wherein the ATP, dATP or dTTP are at a concentration in the range of about 0.1-50 mM.

31. A method according to claim 1, wherein the helicase preparation comprises a single strand binding protein (SSB).

32. A method according to claim 31, wherein the SSB is selected from T4 gene 32 SSB, E. coli SSB, T7 gene 2.5 SSB, phage phi29 SSB and derivatives therefrom.

33. A method according to claim 1, wherein the helicase preparation comprises an accessory protein.

34. A method according to claim 33, wherein the accessory protein for a UvrD helicase is MutL.

35. A method according to claim 1, wherein the helicase preparation comprises E. coli UvrD helicase, ATP, E. coli MutL protein and T4 Gp32.

36. A method according to claim 1, wherein the helicase preparation comprises E. coli RecBCD, ATP, and T4 Gp32 SSB.

37. A method according to claim 1, wherein the helicase preparation comprises T7 Gp4B helicase, dTTP, and T7 Gp2.5 SSB.

38. A method according to claim 1, wherein the helicase preparation comprises the thermostable Tte-UvrD helicase, dATP or ATP.

39. A method according to claim 1, wherein the helicase preparation comprises the thermostable Tte-UvrD helicase, dATP or ATP and a single strand binding protein.

40. A method according to claim 1, wherein step (b) is performed at a substantially single temperature in the range of about 20°

C.-75°

C.

41. A method according to claim 1, wherein step (b) is performed at about 37°

C.

42. A method according to claim 23, wherein step (b) is performed at about 60°

C. and the helicase in the helicase preparation is a thermostable helicase.

43. A method according to claim 1, wherein the target nucleic acid is obtained from a pathogen in a biological sample, and the method further comprises amplifying the target nucleic acid to detect the pathogen.

44. A method according to claim 1, wherein the target DNA is chromosomal DNA and the method further comprises detecting a sequence variation in the chromosomal DNA.

45. A method according to claim 44, wherein the sequence variation is a single nucleotide polymorphism.

46. A method for determining whether a helicase is suited for selectively amplifying a target nucleic acid in a helicase-dependent reaction, comprising;

(a) preparing a helicase preparation comprising the helicase, an NTP or dNTP, a buffer, wherein the buffer has a pH in the range of about pH 6.0-9.0, a concentration of NaCl or KCl in a concentration range of 0-200 mM, and Tris-acetate or Tris-HCl and an accessory protein to which is added one or more SSBs unless the helicase is thermostable wherein the SSB is not required;

(b) adding a target nucleic acid, oligonucleotide primers, four dNTPs and a DNA polymerase to the helicase preparation;

(c) incubating the mixture at a temperature between about 20°

C. and 75°

C.; and

(d) analyzing the DNA to determine whether selective amplification has occurred.

47. A method according to claim 46, further comprising optimizing the conditions of helicase-dependent amplification by varying the concentration of any or all of:

the helicase;

the SSB;

the accessory protein;

the NTP or dNTP;

the salt concentration;

the pH; and

varying the buffer type;

the temperature;

the time of incubation and the length of the target nucleic acid.

48. A method for exponentially and selectively amplifying a target nucleic acid in a helicase-dependent reaction, the method comprising:

(a) providing single strand templates of the target nucleic acid to be amplified;

(b) hybridizing one or more oligonucleotide primer pairs to the templates of step (a);

(c) synthesizing an extension product of the oligonucleotide primers which are complementary to the templates by means of a DNA polymerase to form a duplex;

(d) contacting the duplex of step (c) with a helicase preparation for unwinding the duplex such that the helicase preparation comprises a helicase and an SSB unless the helicase preparation comprises a thermostable helicase wherein the SSB is optional; and

(e) repeating steps (b)-(d) to exponentially and selectively amplify the target nucleic acid in a helicase-dependent reaction such that amplification does not occur in the absence of the helicase as determined from an ethidium bromide stained gel after electrophoresis.