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Nucleic acid amplification method

A nucleic acid and nucleic acid molecule technology, applied in the field of nucleic acid amplification, can solve the problems of cost detection method design and operation, detection sensitivity problems, etc.

Inactive Publication Date: 2009-09-16
TOYO SEIKAN KAISHA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, these known methods still have problems in terms of cost, design and operation of detection methods, detection sensitivity, etc., so new nucleic acid amplification methods and detection methods that fully satisfy these problems are required

Method used

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Examples

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preparation example Construction

[0105] In the method for preparing circular single-stranded DNA of the present invention, the "complementary sequence (of the 5'-terminal region and 3'-terminal region)" is preferably the sequence of the 5'-terminal region and the 3'-terminal region of the linear single-stranded DNA. The sequence of the region is completely complementary to the sequence, but there may be a base pair mismatch as long as it can anneal to the aforementioned linear single-stranded DNA. The number of such base pairs depends on the reaction conditions and the length of the entire region. For example, it may be 1, 2, or 3 in each region, or may be about 1% to 50% of the total bases in each region. However, those skilled in the art can understand that, in order to perform ligation efficiently, it is preferable that the bases at the 5' end and the 3' end that are actually subjected to the ligation reaction by ligase each form a base with the base of the adapter during circularization. base pair.

[01...

Embodiment

[0124] Substances without a company name after the drug name are manufactured by Wako Pure Chemical Industries, Ltd. In addition, unless otherwise specified, the solvent is water.

[0125] (TE buffer composition)

[0126] 4mmol / l Tris-HCl (trishydroxymethylaminomethane) hydrochloric acid

[0127] 1mmol / l ethylenediaminetetraacetic acid disodium dihydrate (EDTA)

[0128] Adjust to pH 8.0

[0129] (1.2% TAE gel for electrophoresis)

[0130] 1.2% agarose

[0131] 4mmol / l Tris-HCl

[0132] 1mmol / l EDTA

[0133] 1mmol / l acetic acid

[0134] (2.0% TAE gel for electrophoresis)

[0135] 2.0% agarose

[0136] 4mmol / l Tris-HCl

[0137] 1mmol / l EDTA

[0138] 1mmol / l acetic acid

example 1

[0140] [Preparation method of target]

[0141] Enzyme preparations and kit preparations shown below were in accordance with the instructions for use unless otherwise specified.

[0142] After pUC19 (Takara Bio Inc.) was treated with the restriction enzyme DraI (Takara Bio Inc.), electrophoresis was performed (100 V, 50 minutes) using a 1.2% TAE gel for electrophoresis using an electrophoresis tank Mupid-ex (Advance).

[0143] Then, stain with nucleic acid staining reagent SYBR GreenI (manufactured by CAMBREX company), cut out the target DNA fragment (about 2kb) from the gel, reclaim with DNA extraction kit NucleoSpin Extract kit (MACHEREY-NAGEL company), form the target preparation solution ( 10mmol / l).

[0144] [Synthesis of circular single-stranded DNA]

[0145] The primers used for circular single-stranded DNA synthesis are listed in Table 1. 43C is a primer for phosphorylation modification at the 5' end, and Bind is a primer for cartridge purification (manufactured by In...

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Abstract

Disclosed is a nucleic acid amplification method which is based on a new principle and enables to amplify a nucleic acid having a specific nucleotide sequence in a simple manner, within a short time and with efficiency. The nucleic acid amplification method comprises the steps of: (a) conducting a DNA polymerase chain reaction by using, as a template, DNA comprising a nucleotide sequence to be amplified and using a primer pair having a nucleotide sequence complementary to the nucleotide sequence to be amplified, thereby producing a linear DNA fragment; and (b) conducting a chain-substituting DNA polymerase chain reaction in a chaining manner by using cyclic single-stranded DNA comprising the same nucleotide sequence as that of at least one of the primer pair as a template and employing the 3'-terminus of the linear DNA fragment produced in step (a) as the replication origin.

Description

technical field [0001] The present invention relates to a nucleic acid amplification method, particularly a nucleic acid amplification method using a combination of a specific primer and circular single-stranded DNA, a method for detecting the presence or absence of a target gene by the aforementioned method, and a detection kit used in the aforementioned method. Background technique [0002] Currently, analysis methods and detection methods based on specific base sequences of target genes are widely used in research institutions, medical institutions, inspection institutions, and various other sites. For example, in the analysis and detection of pathogenic microorganisms and microscopic animals such as pathogenic bacteria, molds, mites and other allergens contained in samples (body fluids or cell sheets) derived from biological sources such as humans and animals, or samples derived from the environment, In the presence of viruses, pollen, etc., a method for detecting the sp...

Claims

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Application Information

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IPC IPC(8): C12N15/09
Inventor 远山将史
Owner TOYO SEIKAN KAISHA LTD
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