Method of amplifying nucleic acid by using double-stranded nucleic acid as template

• US 20030207292A1
• Filed: 04/08/2003
• Published: 11/06/2003
• Est. Priority Date: 04/07/2000
• Status: Active Grant

• Current Assignee: Eiken Kagaku Kabushiki Kaisha
• Original Assignee: Eiken Kagaku Kabushiki Kaisha
• Inventors: Notomi, Tsugunori, Nagamine, Kentaro
• Granted Patent: US 8,017,357 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 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

The present invention provides a nucleic acid synthesis method which involves the step of incubating a double-stranded nucleic acid template under conditions that ensure a complementary strand synthesis reaction using a primer as an origin. This method involves the step of placing a region, to which a primer capable of isothermally amplifying the template nucleic acid will anneal, in a condition that ensures base pairing, using an arbitrary primer. The arbitrary primer initiates the complementary strand synthesis reaction, using the double-stranded nucleic acid as a template and DNA polymerases catalyzing the complementary strand synthesis reaction which comprises the destabilization of the double-stranded nucleic acid and strand displacement, thereby providing a region that can undergo base pairing.

Claims

1. A method for synthesizing nucleic acid using double-stranded nucleic acid as a template, wherein the method comprises:

a) incubating a double-stranded nucleic acid template and an arbitrary primer in the presence of a DNA polymerase that catalyzes a complementary strand synthesis reaction accompanying strand displacement, under a condition that ensures the synthesis of complementary strand using the arbitrary primer as an origin, such that a region of the target template nucleic acid to be annealed by a primer capable of amplifying the template nucleic acid at a constant temperature is placed in a condition that allows the region to undergo base pairing;

b) annealing a primer that can amplify the template nucleic acid at a constant temperature, to the region obtained in the step a), which is placed in a condition such that it can undergo base pairing; and

c) carrying out the complementary strand synthesis using the primer as a synthesis origin.

2. The method according to claim 1, wherein the step a) is carried out in the presence of a melting temperature regulator.

3. The method according to claim 2, wherein the melting temperature regulator is at least one of compounds selected from the group consisting of betaine, proline, dimethyl sulfoxide, and trimethylamine-N-oxide.

4. A method for synthesizing a nucleic acid in which a plural number of nucleotides, constituting a specific region of a double-stranded nucleic acid template that is constituted by complementary nucleotide sequences, are connected on a single-strand, wherein the method comprises:

a) incubating a double-stranded nucleic acid template and an arbitrary primer in the presence of DNA polymerase that catalyzes a complementary strand synthesis reaction accompanying strand displacement under a condition that ensures the synthesis of complementary strand using the arbitrary primer as an origin, such that a region of the target template nucleic acid to be annealed by a second primer, is placed in a condition that allows the region to undergo base pairing;

b) annealing the second primer to the region obtained in the step a), which is placed in a condition such that it can undergo base pairing, and carrying out the complementary strand synthesis using the second primer as an origin, wherein the 3′

-end of the second primer anneals to a region that defines the 3′

-side of one of the strands constituting the specific region and the 5′

-end of the second primer comprises a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as an origin;

c) placing a region of the extended product of the second primer synthesized in the step b), to which a first primer will anneal, in a condition such that the region can undergo base pairing, wherein the 3′

-end of the first primer anneals to a region that defines the 3′

-side of said region in the extended product obtained using the second primer as an origin;

d) annealing the first primer to the region obtained in the step c), which is placed in a condition such that it can undergo base pairing and carrying out the synthesis of complementary strand using the first primer as an origin; and

e) allowing self annealing at the 3′

-end of the extended product of the first primer synthesized in the step d) to occur and carrying out the synthesis of complementary strand using the extended product itself as a template, and obtaining nucleic acid in which a plural number of the nucleotides constituting the specific region are connected on single strand.

5. The method according to claim 4, wherein the arbitrary primer in the step a) is a first primer.

6. The method according to claim 4, wherein the step c) is carried out by displacement according to a complementary strand synthesis reaction using as an origin a fourth primer that anneals to the 3′

-side of the region of the template annealed by the second primer.

7. The method according to claim 4, wherein the step e) further comprises a step of converting the extended product of the first primer into a single strand by displacement according to a complementary strand synthesis reaction using as an origin a third primer that anneals to the 3′

-side of the region of the template annealed by the first primer.

8. The method according to claim 4, wherein the 5′

-end of the first primer comprises a nucleotide sequence complementary to an arbitrary region of the complementary strand synthesis reaction product obtained using the first primer as an origin.

9. A method for amplifying nucleic acid in which a plural number of nucleotides, constituting a specific region of a double-stranded nucleic acid template that is constituted by complementary nucleotide sequences, are connected on a single-strand, wherein the method comprises:

1) allowing self annealing at the 3′

-end of the extended product of the first primer produced by the method according to claim 7 and carrying out the complementary strand synthesis reaction using the extended product as an origin;

2) annealing the second primer or the first primer to a loop region that is formed by self annealing of the 3′

-end and carrying out the synthesis of complementary strand using the primer as an origin;

3) allowing strand displacement of the extended product from the 3′

-end to occur by the complementary strand synthesis reaction of the step

     2) so that the 3′

-end can undergo base pairing;

4) carrying out the complementary strand synthesis reaction using as a template the displaced strand itself obtained in the step

     3), which can undergo base pairing and using its 3′

-end as an origin to displace a complementary strand synthesized in the step

     2) using the loop region as an origin, thereby producing a single-stranded nucleic acid; and

5) repeating the steps

     2) to

     4) to amplify the desired nucleic acid.

10. The method according to claim 9, wherein the method further comprises:

6) carrying out the complementary strand synthesis reaction by self annealing of the 3′

-end of the single-stranded nucleic acid produced in the step

     4);

7) annealing the second primer or the first primer to a loop region that is formed by self annealing of the 3′

-end and carrying out the complementary strand synthesis using the primer as an origin;

8) allowing strand displacement of the extended product from the 3′

-end to occur by the complementary strand synthesis reaction of the step

     7), so that the 3′

-end can undergo base pairing;

9) carrying out the complementary strand synthesis reaction using as a template the displaced strand itself obtained in the step

     8), which can undergo base pairing and using its 3′

-end as an origin to displace a complementary strand synthesized in the step

     7) using the loop region as an origin, thereby producing a single-stranded nucleic acid; and

10) repeating the steps

     7) to

     9) to amplify the desired nucleic acid.

11. A method for detecting a target nucleotide sequence in a sample, the method comprising carrying out the amplification method according to claim 10 and observing whether or not the amplification reaction product has been generated.

12. A method according to claim 11, wherein the method according to claim 10 is carried out in the presence of a nucleic acid detection agent, further comprising determining whether or not the amplification reaction product has been generated based on the signal change of the detection agent.

13. A method for detecting mutation by the detection method according to claim 11, wherein mutation in a nucleotide sequence to be amplified prevents the synthesis of complementary strand, at least at one 3′

-end that is the origin of the complementary strand synthesis which constitutes the amplification method.

14. A method for amplifying a nucleic acid in which a plural number of a nucleotides, constituting a specific region of a template double-stranded nucleic acid which is constituted by complementary nucleotide sequences, are connected on a single-strand, wherein the method comprises a step of incubating the following elements:

a target comprising a double-stranded nucleic acid template comprising a specific region to be amplified;

a DNA polymerase that catalyzes the complementary strand synthesis reaction accompanying strand displacement;

a first primer, the 3′

-end of which anneals to a region that defines the 3′

-side of one of the strands constituting the specific region and the 5′

-end of which comprises a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin;

a second primer, the 3′

-end of which anneals to a region that defines the 3′

-side of one of the strands constituting the specific region and the 5′

-end of which comprises a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin; and

a nucleotide substrate;

under a condition that enables the synthesis of complementary strand using the first primer as an origin.

15. The method according to claim 14, wherein the elements further comprise:

a third primer that becomes an origin of the complementary strand synthesis reaction, using the 3′

-side of the region in the template to be annealed by the first primer as an origin; and

a fourth primer that becomes an origin of the complementary strand synthesis reaction, using the 3′

-side of the region in the template to be annealed by the second primer as an origin.

16. The method according to claim 14, wherein the incubation is carried out in the presence of a melting temperature regulator.

17. The method according to claim 16, wherein the melting temperature regulator is at least one of compounds selected from the group consisting of betaine, proline, dimethyl sulfoxide, and trimethylamine-N-oxide.

18. A method for placing a region of a target template nucleic acid to be annealed by a primer that initiates a reaction of amplifying a template nucleic acid at a constant temperature, in a condition such that the region can undergo base pairing, wherein the method comprises the step of incubating a template double-stranded nucleic acid, an arbitrary primer, and a primer capable of amplifying the template nucleic acid at a constant temperature, in the presence of DNA polymerase, which catalyzes a complementary strand synthesis accompanying strand displacement, under a condition that ensures the synthesis of complementary strand, using the arbitrary primer as an origin.