Nucleic acid amplification method

A technology for amplifying nucleic acids and oligonucleotides, which is applied in biochemical equipment and methods, microbiological measurement/inspection, fermentation, etc. It can solve problems such as long reaction time, increased time consumption, troublesome temperature control, etc., and achieve high The effect of amplification efficiency

Active Publication Date: 2009-02-18
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 1

[0158] Amplification reaction of nucleic acid

[0159] (1) Preparation of nucleic acid sample solution containing target nucleic acid fragments

[0160] After heating 3.0 ng of Human Genomic DNA (manufactured by Clontech) at 98° C. for 3 minutes to make it single-stranded, the sequence in the β-actin gene was amplified under the following conditions.

[0161]

[0162] Primer design was performed using the β-actin gene as a target. The sequences of the respective primers are shown below.

[0163] Primer (1) (forward primer):

[0164] 5'-GGGCATGGGTCAGAAGGATT-3' (SEQ ID NO: 1)

[0165] Primer (2) (reverse primer):

[0166] 5'-CCTCGTCGCCCACATAG-3' (SEQ ID NO: 2)

[0167] The positional relationship of the above primers relative to the β-actin gene is specifically as follows: figure 1 shown.

[0168] Here, the sequence X is 5'-CCCAG-3', and the distance between the two sequences X is 54 bases. Primer (1) is designed for the region between the 5' terminal base of the sequ...

Embodiment 2

[0189] Electrophoresis of amplified products

[0190] Electrophoresis was performed at 100 V for 60 minutes using 3% by weight agarose gel, 0.5 x TBE buffer (50 mM Tris, 45 mM boric acid, 0.5 mM EDTA, pH 8.4). The result is as image 3 shown. The electrophoretic patterns of the 6 samples are substantially the same, thus it can be seen that the amplified products are obtained according to the same reaction mechanism.

Embodiment 3

[0191] Cloning of amplified product

[0192] After electrophoresis, the gel in the region below 200 bp was cut out, and the DNA in the gel was recovered using QIAEX II (manufactured by Qiagen).

[0193] The recovered DNA was recombined into a vector using TOPO TA Cloning Kit (manufactured by Invitrogen), and Escherichia coli was transformed with the vector. The transformed Escherichia coli were cultured in LB medium supplemented with ampicillin.

[0194] Plasmid DNA was recovered from the cultured Escherichia coli using QIAprep Miniprep (manufactured by Qiagen).

[0195]The recovered plasmid DNA was sequenced to determine the base sequence. M13 reverse primer (M13 Reverse Primer) was used as a primer.

[0196] M13 reverse primer

[0197] 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 3)

[0198] From the results of sequencing, it was found that the following three types of amplification products existed.

[0199] (1)

[0200] 5'-GGGCATGGGT CAGAAGGATT CCTATGTGGG CGACGAGG-3'

[02...

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Abstract

The present invention provides a nucleic acid amplification method by using a oligonucleotide primer and a DNA polyase. The nucleic acid amplification method provided by the invention comprises performing incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase having strand displacement activity, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3' end of the primer and thus amplifying the nucleic acid fragment, wherein the characteristic is that a first oligonucleotide primer and a second oligonucleotide primer are designed in such a way that a region which contains two identical sequences X of serial 4 or more nucleotides within the region of 200 or less nucleotides, or a part thereof can be amplified.

Description

technical field [0001] The present invention relates to a method for amplifying nucleic acid. More specifically, the present invention relates to a method for amplifying nucleic acid, which is characterized in that DNA polymerase is used to perform polymerase reaction by incubating the reaction solution. 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 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 the primer (extension step). In a general PCR method, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12Q1/68
CPCC12Q1/6844
Inventor 三好隼人岩木义英森寿弘
Owner FUJIFILM CORP
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