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Gene mutation detection method and fluorescence-labeled oligonucleotide used in same

An oligonucleotide and fluorescent labeling technology, applied in the field of oligonucleotides, can solve the problems of impossible to correctly distinguish signals and difficult on-site application, and achieve the effect of high sensitivity and high precision

Inactive Publication Date: 2017-11-28
NIPPON STEEL & SUMIKIN ECO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, one clamp primer is used for the amplification reaction, but since the detection is performed with SYBR Green, it is impossible to correctly determine whether the signal is derived from the mutant gene, and it is considered difficult to apply to the clinical field where accuracy is required.

Method used

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  • Gene mutation detection method and fluorescence-labeled oligonucleotide used in same
  • Gene mutation detection method and fluorescence-labeled oligonucleotide used in same
  • Gene mutation detection method and fluorescence-labeled oligonucleotide used in same

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Experimental program
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Embodiment approach

[0041] Next, the present invention will be described in further detail with reference to preferred embodiments. In the present invention, DNA, RNA, cDNA, mRNA, rRNA, NTPs, dNTPs, fluorescently labeled oligonucleotides, hybridization, hybridization, intercalator, primer, annealing, extension reaction, thermal denaturation reaction, nucleic acid melting curve, PCR, Terms such as RT-PCR, PCR method using PNA, devices for nucleic acid detection (gene detection), SNP (SNP: single nucleotide polymorphism), etc. are commonly used in molecular biology, genetic engineering, etc. The terms have the same meaning.

[0042] In the present invention, "wild-type gene" refers to a gene that has no variation in the nucleotide sequence and contains genetic information that functions normally. Herein, the genetic information refers not only to the transcriptional region encoding information such as rRNA, mRNA, etc., but also includes regulatory regions of expressed genes such as promoters.

[...

Embodiment 1

[0070] [Example 1] High-sensitivity detection of JAK2 gene variation (model system with PCR product as target)

[0071] As a template for the PCR reaction, the PCR product (363 bp) of a part of the human JAK2 gene sequence is a wild-type gene: the ratio of the mutant gene is 0:100, 90:10, 99:1, 99.5:0.5, 99.9:0.1, The method of 100:0 was set to a total of 10,000 copies / μl and prepared. Each reaction solution contained: 1 μl of template DNA (10000 copies), KOD+DNA polymerase (Toyobo Co., Ltd.) as a DNA polymerase, 4 kinds of dNTPs (0.2 mM each), forward primer (SEQ ID NO: 1. Final concentration 1.0 μM), reverse primer (SEQ ID NO: 2, final concentration 0.2 μM), magnesium sulfate solution (final concentration 1 mM), specified amount of KOD+ polymerase, and the 3' end with carboxy rhodan Ming 6G (CR6G) labeled quencher probe (SEQ ID NO: 3, final concentration 0.05 μM). Furthermore, the quencher probe functions as a clamp primer and a fluorescently-labeled oligonucleotide for de...

Embodiment 2

[0085] [Example 2] High-sensitivity detection of JAK2 gene variation (real sample)

[0086] Genomic DNA was extracted from the blood of a myeloproliferative neoplasm patient and used as a template DNA for PCR. In addition, DNA extraction was performed using QIAamp DNA Mini Kit (Qiagen). Each reaction solution is: PPD mix (Toyobo Co.), forward primer (SEQ ID NO: 1, final concentration 1.2 μM) dissolved in PPD mix, reverse primer (SEQ ID NO: 4, final concentration 0.2 μM) ) and the quenching probe (SEQ ID NO:5, final concentration 0.12 μM) whose 3' end was labeled with carboxyrhodamine 6G (CR6G) mixed solution 2.4 μl, KOD mix (Toyobo Co., Ltd.) 3.6 μl, containing A solution of 30 ng of genomic DNA and 6 μl of sterilized water were prepared to make a total of 12 μl. In addition, the quencher probe functions as a clamp primer and a fluorescently-labeled oligonucleotide for detection of a target nucleic acid in the same manner as in [Example 1].

[0087] In the following sequenc...

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Abstract

Provided is a technique that makes it possible to rapidly and easily detect, with high sensitivity, a mutant gene in a gene group including many wild-type genes. In the present invention, a measurement method is for measuring nucleic acids for the purpose of specifically detecting a genotype that is to be tested for, in genes or specimens for which it is possible that multiple genetic polymorphisms exist. The measurement method is characterized by: hybridizing an oligonucleotide labeled with a fluorescent dye with a genotype other than that tested for, so as to inhibit gene amplification of said genotype; hybridizing the same fluorescence-labeled oligonucleotide as described above with an amplification product derived from the genotype tested for, which was amplified in the same gene amplification step as described above; and specifically detecting the genotype tested for, on the basis of the change in fluorescence intensity of the fluorescent dye before and after hybridization. A fluorescence-labeled oligonucleotide that can be used in the method is also provided.

Description

【Technical field】 [0001] The present invention relates to an oligonucleotide, a method for specifically amplifying nucleic acid using the same, a method for measuring it, and a method for analyzing data obtained by the method. Specifically, it relates to a specific amplification method for suppressing the amplification of a target nucleic acid by carrying out a nucleic acid amplification reaction in a state where an oligonucleotide is hybridized to a target nucleic acid, various nucleic acids using fluorescently-labeled oligonucleotides, etc. Measuring method, various measuring methods related to various nucleic acids based on the principle of measuring the decrease in luminescence of fluorescent dyes before and after hybridization that occurs when fluorescently labeled oligonucleotides are hybridized to target nucleic acids, and oligonucleotides used therein acid. 【Background technique】 [0002] Mutations in genes are becoming one of the causes of cancer. Therefore, as an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09G01N21/64
CPCC12N15/09G01N21/64C12Q1/6858C12Q2549/126C12Q2563/107C12Q2565/107C12Q2525/117C12Q2537/163C12N15/1031C12Q1/6827C12Q2600/156G01N21/6486G01N33/52G01N33/582
Inventor 山口正裕藏田信也小松则夫森下总司
Owner NIPPON STEEL & SUMIKIN ECO TECH
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