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

• US 8,017,357 B2
• Filed: 03/30/2001
• Issued: 09/13/2011
• Est. Priority Date: 04/07/2000
• Status: Active Grant

• Current Assignee: Eiken Kagaku Kabushiki Kaisha
• Original Assignee: Eiken Kagaku Kabushiki Kaisha
• Inventors: Nagamine, Kentaro, Notomi, Tsugunori
• Primary Examiner(s): Kim; Young J
• Assistant Examiner(s): Bertagna; Angela
• Application Number:
  US10/240,460
• Publication Number:
  US 20030207292A1
• Time in Patent Office: 3,819 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/24.33
• US Class Current: 435/91.2
• 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 amplifying a double-stranded nucleic acid template comprising a specific region, wherein the method comprises:

a) initiating incubation of the 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 the complementary strand using the arbitrary primer as an origin without denaturation of the double-stranded nucleic acid template, such that a region on one strand of the double-stranded nucleic acid template located 3′

of the specific region and 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 step a) and carrying out complementary strand synthesis using the second primer as an origin to obtain an extended product, wherein the 3′

-end of the second primer anneals to the region obtained in step a) and the 5′

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

c) placing a region of the extended product of the second primer synthesized in step b), located 3′

of the specific region and to be annealed by a first primer, in a condition such that the region can undergo base pairing, wherein the 3∝

-end of the first primer anneals to the region of the extended product obtained using the second primer as an origin, and 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 3′

end of the second primer;

d) annealing the first primer to the region obtained in step c) and carrying out complementary strand synthesis using the first primer as an origin to obtain an extended product of the first primer;

e) allowing self annealing at the 3′

-end of the extended product of the first primer synthesized in step d) to occur, thereby forming a loop, and carrying out complementary strand synthesis using the extended product of step d) as a template and the 3′

end of the formed loop as an origin to obtain a nucleic acid in which a plurality of copies of the specific region are connected on a single strand, wherein the step 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 3′

end of the first primer,

wherein (i) the displacement in step c) is carried out beginning from where the 3′

end portion of the fourth primer anneals to the region obtained in step a) and continues to at least the portion of the extended product of the second primer to which the first primer anneals, (ii) the displacement in step e) is carried out beginning from where the 3′

end portion of the third primer anneals to the region obtained in step c) and continues to the 3′

end of the extended product of the first primer, or (iii) both (i) and (ii) are carried out; and

further comprising;

f) allowing self annealing at the 3′

-end of the nucleic acid produced by the complementary strand synthesis in step e) to form a loop and carrying out complementary strand synthesis using the 3′

end of the formed loop as an origin and the nucleic acid as a template;

g) annealing the second primer to the loop that is formed in step e) and the first primer to the loop that is formed in step f), and carrying out complementary strand synthesis using the first and second primers as an origin;

h) allowing strand displacement of the extended product of step f) so that the 3′

-end thereof can undergo base pairing to form a loop;

i) carrying out complementary strand synthesis using as a template the displaced strand obtained in step h), and using the loop formed at its 3′

-end as an origin to displace a complementary strand synthesized in step g), thereby producing a single-stranded nucleic acid comprising a plurality of copies of the specific region connected on the single strand; and

j) repeating steps g) to i) to amplify the nucleic acid in which the plurality of copies of the specific region are connected on the single strand.

2. The method according to claim 1, wherein the method further comprises:

k) allowing self annealing of the 3′

-end of the single-stranded nucleic acid produced in step i) to form a loop;

l) annealing the first primer to the loop that is formed in step k) and carrying out complementary strand synthesis using the first primer as an origin;

m) allowing strand displacement of the extended product of step l), so that the 3′

-end can undergo base pairing to form a loop;

n) carrying out complementary strand synthesis using as a template the displaced strand obtained in step m) and using the loop at its 3′

-end as an origin to displace a complementary strand synthesized in the step l), thereby producing a single-stranded nucleic acid; and

o) repeating steps l) to n) to amplify the nucleic acid in which a plurality of copies of the specific region are connected on a single strand.

3. A method for detecting a target nucleotide sequence in a sample, the method comprising carrying out the amplification method according to claim 1 and observing whether or not the amplification reaction product has been generated.

4. The method according to claim 3, wherein the method is carried out in the presence of a nucleic acid detection agent, the method further comprising determining whether or not the amplification reaction product has been generated based on the signal change of the detection agent.

5. A method for detecting mutation of a target nucleotide sequence by amplification, said method comprising:

a) initiating incubation of a double-stranded nucleic acid template comprising a specific region 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 the complementary strand using the arbitrary primer as an origin without denaturation of the double-stranded nucleic acid template, such that a region on one strand of the double-stranded nucleic acid template located 3′

of the specific region and 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 step a) and carrying out complementary strand synthesis using the second primer as an origin to obtain an extended product, wherein the 3′

-end of the second primer anneals to the region obtained in step a) and the 5′

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

c) placing a region of the extended product of the second primer synthesized in step b), located 3∝

of the specific region and to be annealed by a first primer, in a condition such that the region can undergo base pairing, wherein the 3′

-end of the first primer anneals to the region of the extended product obtained using the second primer as an origin, and 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 3′

end of the second primer;

d) annealing the first primer to the region obtained in step c) and carrying out complementary strand synthesis using the first primer as an origin to obtain an extended product of the first primer;

e) allowing self annealing at the 3′

-end of the extended product of the first primer synthesized in step d) to occur, thereby forming a loop, and carrying out complementary strand synthesis using the extended product of step d) as a template and the 3′

end of the formed loop as an origin to obtain a nucleic acid in which a plurality of copies of the specific region are connected on a single strand, wherein the step 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 3′

end of the first primer,

wherein (i) the displacement in step c) is carried out beginning from where the 3′

end portion of the fourth primer anneals to the region obtained in step a) and continues to at least the portion of the extended product of the second primer to which the first primer anneals, (ii) the displacement in step e) is carried out beginning from where the 3′

end portion of the third primer anneals to the region obtained in step c) and continues to the 3′

end of the extended product of the first primer, or (iii) both (i) and (ii) are carried out; and

further comprising;

f) allowing self annealing at the 3′

-end of the nucleic acid produced by the complementary strand synthesis in step e) to form a loop and carrying out complementary strand synthesis using the 3′

end of the formed loop as an origin and the nucleic acid as a template;

g) annealing the second primer to the loop that is formed in step e) and the first primer to the loop that is formed in step f), and carrying out complementary strand synthesis using the first and second primer as an origin;

h) allowing strand displacement of the extended product of step f) so that the 3′

-end can undergo base pairing to form a loop;

i) carrying out complementary strand synthesis using as a template the displaced strand obtained in step h) and using the loop at its 3′

-end as an origin to displace a complementary strand synthesized in step g), thereby producing a single-stranded nucleic acid;

j) repeating steps g) to i) to amplify the nucleic acid in which a plurality of copies of the specific region are connected on a single strand;

k) allowing self annealing of the 3′

-end of the single-stranded nucleic acid produced in step i) to form a loop;

l) annealing the first primer to the loop that is formed in step k) and carrying out complementary strand synthesis using the first primer as an origin;

m) allowing strand displacement of the extended product of step l), so that the 3′

-end can undergo base pairing to form a loop;

n) carrying out complementary strand synthesis using as a template the displaced strand obtained in step m) and using the loop at its 3′

-end as an origin to displace a complementary strand synthesized in step l) using the loop region as an origin, thereby producing a single-stranded nucleic acid;

o) repeating steps l) to n) to conduct an amplification reaction; and

p) observing whether or not an amplification reaction product has been generated;

wherein mutation in a nucleotide sequence to be amplified prevents the synthesis of the complementary strand at least at one 3′

-end that is an origin of complementary strand synthesis.