Isothermal, magnetic particle-mediated acid amplification

• US 5,545,540 A
• Filed: 01/19/1995
• Issued: 08/13/1996
• Est. Priority Date: 06/09/1993
• Status: Expired due to Term

• Current Assignee: Tecan Trading AG (Tecan Group AG)
• Original Assignee: Gamera Bioscience Corporation (Tecan Group AG)
• Inventors: Mian, Alec
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Schreiber, David
• Application Number:
  US08/375,226
• Time in Patent Office: 572 Days
• Field of Search: 435/91.2, 435/6
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides a process for amplification of specific nucleic acid sequences based upon the separation of nucleic acid strands by an electromagnetic field. This means of separation allows the use of mesophilic polymerases in the amplification process, thereby increasing the speed and fidelity of the amplification process.

Claims

1. A method of amplifying a specific single-stranded target nucleic acid, the method comprising the steps of:

(a) incorporating into a nucleic acid strand complementary to the target nucleic acid a solid phase primer to create a solid phase strand bound to the target nucleic acid;

(b) separating the solid phase strand and the target nucleic acid;

(c) incorporating into a nueleic acid strand complementary to the solid phase strand a magnetic primer to yield a duplex having one solid phase strand and one magnetic strand;

(d) separating the solid phase strand from the magnetic strand by applying an electromagnetic field, wherein application of the electromagnetic field provides a motive force on the magnetic strand of sufficient strength to physically separate the two strands, resulting in dissociation of the duplex;

(e) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;

(f) extending the annealed primers with a suitable DNA polymerase; and

(g) repeating steps (d) through (f) as many times as necessary to obtain a desired quantity of amplified DNA.

4. The method according to claim 1, wherein the magnetic primer is an oligonucleotide bound to a ferritin molecule.

5. The method according to claim 1, wherein the magnetic primer is an oligonucleotide bound to a paramagnetic bead.

6. The method according to claim 1, wherein the solid phase primer is an oligonucleotide bound to a glass matrix.

13. The method according to claim 1, wherein the method takes place in a buffer consisting essentially of:

(a) from 10 to 20 mM Tris-HCl (pH 7-8);

(b) from 5 to 15 mM MgCl₂ ;

(c) from 0 to 5 mM DTT;

(d) from 0.1 to 0.3 mM each of 4 dNTPs;

(e) from 0.1 to 0.5 mM of either a solid phase primer or a magnetic primer;

(f) from 0 to 5.6M betaine; and

(g) target nucleic acid.

2. A method of amplifying a specific single-stranded target nucleic acid, the method comprising the steps of:

(a) incorporating into a nucleic acid strand complementary to the target nucleic acid a magnetic primer to create a magnetic strand bound to the target nucleic acid;

(b) separating the magnetic strand and the target nucleic acid;

(c) incorporating into a nucleic acid strand complementary to the magnetic strand a solid phase primer to yield a duplex having one solid phase strand and one magnetic strand;

(d) separating the solid phase strand from the magnetic strand by applying an electromagnetic field, wherein application of the electromagnetic field provides a motive force on the magnetic strand of sufficient strength to physically separate the two strands, resulting in dissociation of the duplex;

(e) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;

(f) extending the annealed primers with a suitable DNA polymerase; and

(g) repeating steps (d) through (f) as many times as necessary to obtain a desired quantity of amplified DNA.

7. The method according to claim 2, wherein the magnetic primer is an oligonucleotide bound to a ferritin molecule.

8. The method according to claim 2, wherein the magnetic primer is an oligonucleotide bound to a paramagnetic bead.

9. The method according to claim 2, wherein the solid phase primer is an oligonucleotide bound to a glass matrix.

14. The method according to claim 2, wherein the method takes place in a buffer consisting essentially of:

(a) from 10 to 20 mM Tris-HCl (pH 7-8);

(b) from 5 to 15 mM MgCl₂ ;

(c) from 0 to 5 mM DTT;

(d) from 0.1 to 0.3 mM each of 4 dNTPs;

(e) from 0.1 to 0.5 mM of either a solid phase primer a magnetic primer;

(f) from 0 to 5.6M betaine; and

(g) target nucleic acid.

3. A method of amplifying a specific double stranded target nucleic acid, the method comprising the steps of:

(a) separating the strands of the target nucleic acid to yield a first strand and a second strand;

b) incorporating a solid phase primer into a strand complementary to the first strand to yield a first DNA duplex having the first strand and a solid phase strand, and incorporating a magnetic primer into a strand complementary to the second strand to yield a second DNA duplex having the second strand and a magnetic strand;

(c) separating the strands of the first DNA duplex and of the second DNA duplex;

(d) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;

(f) extending the annealed primers with a suitable DNA polymerase;

(g) separating the solid phase strand from the magnetic strand by applying an electromagnetic field, wherein application of the electromagnetic field provides a motive force on the magnetic strand of sufficient strength to physically separate the two strands, resulting in dissociation of the duplex; and

(h) repeating steps (c) through (g) as many times as necessary to obtain a desired quantity of amplified DNA.

10. The method according to claim 3, wherein the magnetic primer is an oligonucleotide bound to a ferritin molecule.

11. The method according to claim 3, wherein the magnetic primer is an oligonucleotide bound to a paramagnetic bead.

12. The method according to claim 3, wherein the solid phase primer is an oligonucleotide bound to a glass matrix.

15. The method according to claim 3, wherein the method takes place in a buffer consisting essentially of:

(a) from 10 to 20 mM Tris-HCl (pH 7-8);

(b) from 5 to 15 mM MgCl₂ ;

(c) from 0 to 5 mM DTT;

(d) from 0.1 to 0.3 mM each of 4 dNTPs;

(e) from 0.1 to 0.5 mM of either a solid phase primer or a magnetic primer;

(f) from 0 to 5.6M betaine; and

(g) target nucleic acid.