Helicase-dependent amplification of nucleic acids

• US 20060154286A1
• Filed: 01/05/2006
• Published: 07/13/2006
• Est. Priority Date: 09/20/2002
• Status: Active Grant

• Current Assignee: BioHelix Corp. (Quidel Corporation)
• Original Assignee: New England Biolabs Incorporated
• Inventors: Tang, Wen, Xu, Yan, Kong, Huimin, Vincent, Myriam
• Granted Patent: US 7,662,594 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

Claims

1. A method for identifying an RNA molecule, the method comprising:

(a) synthesizing a cDNA from the RNA molecule by reverse transcription (RT) to form an RNA/DNA duplex;

(b) at least partially separating the duplex into a cDNA and an RNA using a thermostable helicase;

(c) amplifying the cDNA ; and

(d) characterizing the cDNA to determine the identity of the RNA molecule

2. A method according to claim 1, wherein the RT in step (a) results from a reverse transcriptase or a DNA polymerase with RT activity.

3. A method according to claim 1, wherein step (c) further comprises amplifying the cDNA by helicase-dependent amplification, loop-mediated isothermal amplification, rolling cycle amplification, strand-displacement amplification or polymerase chain reaction.

4. A method according to claim 1, wherein a cDNA is synthesized from the RNA from step (b) according to step (a).

5. A method according to claim 1, wherein step (a), step (b) and step (c) occur in separate buffers or reaction mixtures.

6. A method according to claim 1, wherein step (a) and step (b) or step (b) and step (c) occur in one buffer.

7. A method according to claim 1, wherein step (a), step (b) and step (c) occur in one buffer.

8. A method according to claim 1, wherein the thermostable helicase has an effective activity in the absence of a single strand binding protein.

9. A method according to claim 1 wherein step (b) further comprises using the thermostable helicase in a reaction mixture that is essentially free of a single strand binding protein.

16. A method according to claim 1 or 15, wherein amplification is isothermal.

17. A method according to claim 1 or 15, wherein steps (b)-(e) are performed at a substantially single temperature in the range of about 20°

C.-75°

C.

18. A method according to claim 1 or 15, wherein the target nucleic acid is obtained from a pathogen in a biological sample, and step (e) further comprises amplifying the target nucleic acid to detect the pathogen.

10. A method for exponentially and selectively amplifying a target nucleic acid, the method comprising:

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

(b) adding oligonucleotide primers for hybridizing 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 thermostable helicase for unwinding the duplex in a reaction mixture that is essentially free of a single strand binding protein; and

(e) repeating steps (b)-(d) to exponentially and selectively amplify the target nucleic acid.

11. A kit, comprising:

a thermostable helicase, a reverse transcriptase, a DNA polymerase and a buffer containing at least three reagents selected from the group consisting of;

a magnesium salt, a sodium salt, dNTP and dATP;

the kit containing instructions for amplifying a DNA in a reaction that does not utilize a single strand binding protein.

12. A kit according to claim 11, wherein the pH of the buffer is between pH 8 and pH 10.

13. A kit according to claim 11 wherein the polymerase is Bst polymerase.

14. A kit according to claim 11, wherein the thermostable helicase is Tte-UvrD helicase.

15. A method for exponentially and selectively amplifying a target nucleic acid, the method comprising:

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

(b) adding oligonucleotide primers for hybridizing 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; and

(e) repeating steps (b)-(d) to exponentially and selectively amplify the target nucleic acid.