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Method for amplifying nucleic acid

A technology for amplifying nucleic acid and nucleic acid, which is applied in the field of amplifying nucleic acid, and can solve problems such as uncontrollable and difficult pollution

Active Publication Date: 2016-05-11
SYSMEX CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the isothermal amplification, once the reaction starts, the strand displacement reaction is continuously carried out, which cannot be controlled.
Therefore, it is difficult to deal with contamination in the amplification reaction.

Method used

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  • Method for amplifying nucleic acid
  • Method for amplifying nucleic acid
  • Method for amplifying nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0022] In the nucleic acid amplification method of the present embodiment (hereinafter also simply referred to as "method"), by using a photoresponsive nucleic acid, the amplification reaction of the double-stranded nucleic acid can be controlled by light irradiation without going through temperature cycling as in the PCR method. conduct. That is, in the method of this embodiment, the heating step for denaturing and dissociating the double-stranded nucleic acid composed of the template nucleic acid and the complementary strand, and the cooling step for bringing the primer and the template nucleic acid into complementary alignment are not necessary. . Therefore, it is sufficient to incubate the reaction system at a substantially constant temperature. That is, the method of this embodiment can be performed under substantially isothermal conditions.

[0023] The reaction system refers to a limited place or space in which factors necessary for a nucleic acid amplification reacti...

Embodiment 1

[0081] [Example 1: Verification of Light Irradiation Dependence of Nucleic Acid Amplification Reaction Using Photoresponsive Nucleic Acid]

[0082] In this example, it was evaluated whether a light-responsive nucleic acid can be used to perform a nucleic acid amplification reaction dependent on light irradiation.

[0083] 【(1) Preparation of Nucleic Acids and Reagents】

[0084] As a light-responsive nucleic acid, Tsukuba OligoService Co., Ltd. was commissioned to synthesize a single-stranded DNA in which one 2',6'-dimethylazobenzene was bonded every two bases. The sequence of this light-responsive nucleic acid is shown next.

[0085] 5'-CT(Z)TT(Z)AA(Z)GA(Z)AG(Z)GA(Z)GA(Z)TA(Z)TA(Z)CC(Z)TG(Z)AG(Z) )TG(Z)AT(Z)CT(Z)AG(Z)TG(Z)TA(Z)CT(Z)TA-3' (SEQ ID NO: 1)

[0086] In the above sequence, (Z) represents the insertion position of 2',6'-dimethylazobenzene. In addition, 2',6'-dimethylazobenzene is bound to a side chain moiety to become a nucleic acid via D-threoninol inserted into...

Embodiment 2

[0109] [Example 2: Verification of dependence of nucleic acid amplification reaction using photoresponsive nucleic acid on number of light irradiations]

[0110]In this example, it was verified whether a nucleic acid amplification reaction using a photoresponsive nucleic acid proceeds depending on the number of times of light irradiation.

[0111] 【(1) Preparation of Nucleic Acids and Reagents】

[0112] In this example, the same photoresponsive nucleic acid, template DNA, first primer, and second primer as those in Example 1 above were used. Also, in the same manner as in Example 1, PLL-g-Dex (molecular weight of PLL: 8000, grafting rate: 90%) was used as the strand exchange promoting substance, and BstDNA polymerase (New England Biolab) was used as the DNA synthetase. In addition, the reaction solution was used by diluting the attached 10×ThermoPol buffer (New England Biolab).

[0113] 【(2) Nucleic Acid Amplification】

[0114] Prepare 4 buffer solutions containing 1×Thermo...

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Abstract

The present invention relates to a method for amplifying a nucleic acid. More specifically, the present invention relates to a method for amplifying a nucleic acid in a light irradiation dependent manner under a substantially isothermal condition using a photo-responsive nucleic acid.

Description

【Technical field】 [0001] The present invention relates to methods of amplifying nucleic acids. 【Background technique】 [0002] The PCR (polymerase chain reaction) method is the most widely used and widely used nucleic acid amplification method in various fields such as research and medicine. In the PCR method, a nucleic acid is amplified by repeating denaturation of a double-stranded nucleic acid, synthesis of an extended strand from a primer, and the like by repeating temperature cycles of heating and cooling of a reaction solution. In addition, in recent years, a method for amplifying nucleic acid at a constant temperature (approximately 60 to 70° C.) without temperature cycling has been developed. A representative example of such a method includes the LAMP (loop-mediated constant temperature amplification) method (see US Pat. No. 6,410,278). In the LAMP method, a reaction is started by binding primers to a template nucleic acid under a certain temperature condition, and...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12Q1/6844C12Q1/6848C12Q2523/305C12Q2523/319C12Q2531/119C12Q1/6853
Inventor 铃木誓吾桐村浩哉浅沼浩之
Owner SYSMEX CORP
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