Method of amplifying template DNA molecule using strand-displacing DNA polymerase capable of carrying out isothermal amplification

• US 20050277146A1
• Filed: 05/27/2005
• Published: 12/15/2005
• Est. Priority Date: 05/28/2004
• Status: Abandoned Application

• Current Assignee: Aisin Seiki Co., Ltd. (Aisin Corp.), Riken
• Original Assignee: Aisin Seiki Co., Ltd. (Aisin Corp.), Riken
• Inventors: Shibata, Takehiko, Shigemori, Yasushi, Mikawa, Tsutomu
• Application Number:
  US11/138,443
• Publication Number:
  US 20050277146A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

A method of amplifying a template DNA molecule in an isothermal reaction that can reduce background noise is provided. A method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification includes a step of conducting an amplification reaction with the addition of a single-strand DNA binding protein (SSB) from an extreme thermophile.

Claims

1. A method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification, wherein the amplification reaction is carried out by the addition of a single-strand DNA binding protein (SSB) obtained from an extreme thermophile.

2. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 1, wherein the strand-displacing DNA polymerase is φ

29DNA polymerase.

3. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 1, wherein the extreme thermophile is Thermus thermophilus HB8.

4. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 2, wherein the extreme thermophile is Thermus thermophilus HB8.

5. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 3, wherein the SSB of Thermus thermophilus HB8 has a protein concentration in a range of 0.1 to 0.4 μ

g/μ

L.

6. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 4, wherein the SSB of Thermus thermophilus HB8 has a protein concentration in a range of 0.1 to 0.4 μ

g/μ

L.

7. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 1, wherein the amplification reaction is a rolling circle amplification (RCA) reaction.

8. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 1, wherein the template DNA molecule is a circular DNA molecule.

9. A method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification, comprising the steps of:

annealing a primer to the template DNA molecule; and

extending a complementary strand of the template DNA at the annealed primer as a replication origin by the strand-displacing DNA polymerase, wherein the strand-displacing DNA polymerase is made to act on the template DNA molecule that was annealed with the primer in the above-mentioned annealing step, in which, when the extension reaction portion comes into contact with an already-synthesized complementary strand portion, the extension reaction proceeds while tearing the already-synthesized strand off by the strand-displacing activity,

wherein an SSB obtained from an extreme thermophile is added in the extension step.

10. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the strand-displacing DNA polymerase is φ

29DNA polymerase.

11. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the extreme thermophile is Thermus thermophilus HB8.

12. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 11, wherein the SSB of Thermus thermophilus HB8 has a protein concentration in a range of 0.1 to 0.4 μ

g/μ

L.

13. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the amplification reaction is a rolling circle amplification (RCA) reaction.

14. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the template DNA is a circular DNA molecule.

15. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the extension step is carried out in an isothermal temperature range with a temperature variation of 10°

C. or less.

16. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the extension step is carried out in a temperature range of 60°

C. or less.

17. The method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification according to claim 9, wherein the extension step is carried out in a temperature range of 25 to 42°

C.