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Highly sensitive mutated gene detection method

A technology for mutant genes and detection methods, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc.

Inactive Publication Date: 2013-03-06
MITSUBISHI CHEM MEDIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, there have also been cases of death due to side effects such as interstitial pneumonia

Method used

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  • Highly sensitive mutated gene detection method
  • Highly sensitive mutated gene detection method
  • Highly sensitive mutated gene detection method

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Experimental program
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Embodiment approach 1

[0065] Embodiment 1 will be described. This embodiment mainly relates to claim 2 . The summary of this embodiment is a method for detecting a mutant gene, which is a method for detecting the presence or absence of a known mutant gene mixed in a gene pool, in which all or part of a target site having a sequence of a wild-type gene is hybridized with A clamp primer composed of PNA, a mutation probe that hybridizes to all or part of the target site having the sequence of the mutated gene and at least a part of the sequence is composed of LNA, coexists with the aforementioned gene library in the reaction solution for gene amplification, and passes the gene The amplification method selectively amplifies a detection region including a target site of a mutated gene, thereby detecting the presence or absence of the mutated gene.

[0066] Requirements of Embodiment 1 will be described. First of all, in the present invention, the definitions of terms such as DNA, RNA, nucleic acid, ge...

Embodiment approach 2

[0093] Embodiment 2 will be described. This embodiment mainly relates to claim 3 . The summary of this embodiment is a method for detecting a mutant gene, which is a method for detecting the presence or absence of a known mutant gene mixed in a gene pool, in which all or part of a target site having a sequence complementary to a wild-type gene is used A clamp primer composed of hybridized PNA, a mutation probe that hybridizes to all or a part of the target site having a complementary sequence of the mutated gene and at least a part of the sequence is composed of LNA, coexists with the aforementioned gene library in a reaction solution for gene amplification, The presence or absence of the mutated gene is detected by selectively amplifying the detection region including the target site of the mutated gene by a gene amplification method.

[0094] That is, the aforementioned Embodiment 1 uses the sequence on the sense side of the gene to be detected as a template to detect a mut...

Embodiment approach 3

[0097] Embodiment 3 will be described. This embodiment mainly relates to claim 4 . The summary of this embodiment is a method for detecting a mutant gene, wherein, based on the method of Embodiment 1 or 2 above, the gene amplification method is a PCR method. In this embodiment, descriptions of the same requirements and the like as those in the first embodiment will be omitted, and only the characteristic requirements and the like of this embodiment will be described here.

[0098] The "PCR method" in the present invention includes not only the PCR method based on the most fundamental principle, but also a modified method of the PCR method developed based on it. For example, it corresponds to nested PCR method, RT-PCR method, and the like.

[0099] The conditions of the reaction solution for gene amplification of this embodiment are the same as those of the reaction solution for gene amplification of Embodiment 1, and therefore description thereof is omitted here, and the cha...

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Abstract

Disclosed are various highly sensitive detection methods, particularly improved PNA-LNA-PCR clamp methods, as methods for detecting the presence or absence of a mutated gene included in a gene pool rapidly, in a simple manner, with high accuracy, and with high sensitivity. As a previous step for the main detection step, a previous amplification step is carried out, wherein the previous amplification step comprises allowing (1) a clamp primer which comprises a PNA that can hybridize with the entire region or a part of a target site comprising the sequence for wild-type gene or a sequence complementary to the wild-type gene, (2) a primer which can amplify a region containing a target site comprising the sequence for the mutated gene, and (3); the gene pool to coexist in a reaction solution for a gene amplification reaction and selectively amplifying the region containing the target site of the mutated gene by a gene amplification method.

Description

technical field [0001] The present invention relates to methods for detecting known mutant genes interspersed with wild-type gene clusters. Background technique [0002] Cancer is one of the three major lifestyle-related diseases alongside heart disease and cerebrovascular disease, and is currently the leading cause of death in Japan. Cancer usually occurs when a part of the cells that make up the human body become cancerous and become cancer cells. In most cases, canceration of cells is due to mutations in genes related to cell division, cell proliferation, and the like. Most of the gene mutations that cause cancer are point mutations in which one base is replaced by another base in the gene base sequence. [0003] For early treatment of cancer, it is important to detect cancer cells early. The presence or absence of cancer cells is usually diagnosed under a microscope by a cytologist or the like after collecting pathological tissue sections or cells from a specimen, fix...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q2600/156C12N15/09C12Q1/68C12Q1/6886C12Q2600/106C12Q1/6858C12Q2525/107C12Q2525/113C12Q2537/159C12Q2537/163C12Q1/686
Inventor 松本英郎大出明松田耕一郎藤本英也
Owner MITSUBISHI CHEM MEDIENCE
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