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Nucleic acid amplification method

A nucleic acid and oligonucleotide technology, applied in the field of nucleic acid amplification, can solve problems such as long reaction time, troublesome temperature control, and increased time consumption

Active Publication Date: 2009-01-28
FUJIFILM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the nucleic acid amplification reaction is performed at three different temperatures, temperature control is troublesome, and there is also a problem that the time consumed increases proportionally with the number of cycles
However, the method described in JP 11-509406 Gazette has the problem of requiring relatively long reaction time.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0109] The second embodiment of the present invention comprises the following steps: (a) annealing one of the present at least two primers to a nucleic acid strand serving as a template, through the action of the above-mentioned strand displacement type DNA polymerase, from The 3' end of the primer starts to undergo a synthesis reaction, thereby synthesizing an extension product; (b) applying a higher temperature than that in step (a) to the double-stranded nucleic acid obtained by the above-mentioned process without causing the above-mentioned double-stranded In the case of denaturation, the same oligonucleotide primer used in the step (a) is used to infect the double strands, and the synthesis reaction is carried out from the 3' end of the primer by the action of the above-mentioned strand-displacing DNA polymerase , thereby synthesizing an extension product; (c) annealing a primer different from the primer used in the step (a) to the nucleic acid dissociated in the step (b) ...

Embodiment 1

[0167] Amplification of target nucleic acid sequence in human gene

[0168] (1) Preparation of nucleic acid sample solution containing target nucleic acid fragments

[0169] 7.5 ng of Human Genomic DNA (manufactured by Clontech) was heated at 98° C. for 3 minutes, and a specific sequence in the target nucleic acid was amplified under the following conditions. In addition, as a negative control, a sample was prepared by heating purified water under the same conditions as above.

[0170]

[0171] As primers, the following four primers were designed and purchased from Opelon Corporation. The sequences of the respective primers are shown below. Also, primers (1) and (2) are complementary to sequences in the β-actin gene, and primers (3) and (4) are complementary to sequences in the β2AR gene.

[0172] Primer (1):

[0173] 5'-GGGCATGGGTCAGAAGGATT-3' (SEQ ID NO: 1)

[0174] Primer (2):

[0175] 5'-CCTCGTCGCCCACATAG-3' (SEQ ID NO: 2)

[0176] Primer (3):

[0177] 5'-CTTGCT...

Embodiment 2

[0225] Influence of the concentration of surfactant

[0226] (1) Preparation of nucleic acid sample solution containing target nucleic acid fragments

[0227] 7.5 ng of Human Genomic DNA (manufactured by Clontech) was heated at 98° C. for 3 minutes, and a specific sequence in the target nucleic acid was amplified under the following conditions. In addition, as a negative control, a sample was prepared by heating purified water under the same conditions as above.

[0228]

[0229] As primers, primer (1) and primer (2) used in Example 1 were used.

[0230] Primer (1):

[0231] 5'-GGGCATGGGTCAGAAGGATT-3' (SEQ ID NO: 1)

[0232] Primer (2):

[0233] 5'-CCTCGTCGCCCACATAG-3' (SEQ ID NO: 2)

[0234]

[0235] Tween 20 (Formula 1) was used as surfactant.

[0236] (2) Nucleic acid amplification reaction

[0237] Using a reaction solution having the composition shown below, the amplification reaction was carried out at 60°C for 60 minutes. The enzyme used was Bst. Polymerase...

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Abstract

The invention provides a DNA polymerase using oligonucleotide primer and having strand displacement ability, which is capable of amplifying nucleic acid under substantially constant temperature condition. The invention provides a method of amplifying nucleic acid, comprising: under the substantially constant temperature condition, incubating reaction solution comprising at least a deoxy nucleoside triphosphate, at least a DNA polymerase having strand displacement ability, bivalent catio, at least over 0.01% surfactant, at least two oligonucleotide primers and as nucleic acid segment of templet; the polymerase reaction is carried out by taking 3'-back end of the primer as the start, thereby amplifying the nucleic acid segment.

Description

technical field [0001] The invention relates to a nucleic acid amplification method. More specifically, the present invention relates to a nucleic acid amplification method characterized in that the polymerase reaction is carried out by incubating the reaction solution at a substantially constant temperature using a DNA polymerase having strand displacement ability. Background technique [0002] In the research of molecular biology, generally speaking, amplification of nucleic acid is carried out by enzymatic methods using DNA polymerase. As a nucleic acid amplification method, polymerase chain reaction (PCR) is a well-known method. In order to amplify the target nucleic acid sequence, the PCR method consists of the following three steps: a step of denaturing double-stranded DNA as a template into single-stranded DNA (denaturation step); and a step of annealing primers to single-stranded DNA (annealing step); and, a step of extending the complementary strand starting from ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
CPCC12Q1/6853C12Q2531/119C12Q2527/125C12Q2521/101
Inventor 三好隼人岩木义英森寿弘
Owner FUJIFILM CORP
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