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Method for simultaneously detecting gene mutations of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 2

A technology for polymorphism detection and gene polymorphism, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve the problems of requiring manpower, inability to implement arbitrary sequences, and inability to measure, etc. Achieve the effect of simple operation and easy automatic operation

Inactive Publication Date: 2012-10-31
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are the following problems: (1) one reaction system (one test tube) is required for each mutation related to ethanol metabolism, etc., which requires manpower, cost and time; (2) it is necessary to use DNA requires manpower, cost and time, and cannot be easily measured; (3) Since the amplification product needs to be processed when performing a microarray, there is a risk that the amplification product will be mixed with other samples; etc.
In addition, these methods have the problem that they cannot be implemented for all arbitrary sequences

Method used

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  • Method for simultaneously detecting gene mutations of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 2
  • Method for simultaneously detecting gene mutations of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 2
  • Method for simultaneously detecting gene mutations of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1 (using ALDH2 template oligonucleotide as the detection target and using a single probe)

[0150] Based on the base sequence (sequence number 1 (wild type)) of the rs671 site containing the ALDH2 gene polymorphism, a probe containing C at the 3'end as shown in Table 1 (and wild type (sequence) Nos. 3 and 5) and mutant (corresponding to sequence number 4) and probes containing C at the 5'end (corresponding to wild type (sequence number 6)). In Table 1, for the wild type, the position of the probe indicates the base number in the base sequence shown in SEQ ID NO:1, and for the mutant type, it indicates the base in the base sequence shown in SEQ ID NO: 2. Base number. The P at the 3'end indicates phosphorylation. Mark by TAMRA according to common methods.

[0151] In addition, template oligonucleotides (wild-type sense strand (SEQ ID NO: 9), wild-type antisense strand (SEQ ID NO: 11), mutant-type sense strand (SEQ ID NO: 10), wild-type antisense strand ( The sequen...

Embodiment 2

[0166] Example 2 (The case of using a single probe with ADH2 template oligonucleotide as the detection target)

[0167] Based on the base sequence (sequence number 13 (wild type)) of the rs1229984 site containing the gene polymorphism of ADH2, a probe containing C at the 3'end shown in Table 3 (and wild type (sequence number) 16)) and mutants (corresponding to sequence numbers 17 and 18). In Table 3, for the wild type, the position of the probe indicates the base number in the base sequence shown in SEQ ID NO: 13, and for the mutant type, the position of the probe indicates the base sequence shown in SEQ ID No. 14 The base number in. Mark by BODIPY FL according to common methods.

[0168] In addition, the sequences of template oligonucleotides (corresponding to the wild type (SEQ ID NOs: 21 and 23) and mutant type (corresponding to the sequence numbers 22 and 24)) used as the detection target are shown in Table 3. In Table 3, the position of the oligonucleotide represents the ba...

Embodiment 3

[0186] Example 3 (using buccal swabs, whole blood or purified DNA as the test object and using multiple probes)

[0187] As described below, the following primers are used to amplify the polymorphic regions of buccal swabs, whole blood, or purified DNA by PCR. The probes shown in SEQ ID NO: 4 and 16 were used for Tm analysis.

[0188] First, based on the base sequence (sequence number 1 (wild type)) of the rs671 site containing the ALDH2 gene polymorphism, the primers shown in Table 5 that can amplify the polymorphism site were designed. In Table 5, the position of the probe indicates the base number in the base sequence shown in SEQ ID NO:1.

[0189] In addition, based on the base sequence (sequence number 13 (wild type)) of the rs1229984 site containing the gene polymorphism of ADH2, the primers shown in Table 5 that can amplify the polymorphic site were designed. In Table 5, the position of the primer indicates the base number in the base sequence shown in SEQ ID NO: 13.

[0190]...

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Abstract

The invention relates to a method for simultaneously detecting gene mutations of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 2. Concretely, a probe effective for detection of genetic polymorphism of the ALDH2 gene rs671 and the ADH2 gene rs1229984, a method for simultaneously detecting gene mutations and a kit for the purpose are provided. Thus the invention provides a probe for polymorphism detection, namely a probe for detection of at at least one type of genetic polymorphism of the ALDH2 gene rs671 and the ADH2 gene rs1229984, characterized in that: the probe comprises at least 1 type of fluorescently labeled oligonucleotide selected from (P1) to (P3') defined in the description.

Description

Technical field [0001] The present invention relates to a method for detecting acetaldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 2 (ADH2) gene mutations, and nucleic acid probes and kits used in the detection method. Background technique [0002] Representative enzymes related to ethanol metabolism are ADH (Alcohol ehydrogenase) and ALDH (Aldehyde dehydrogenase). Among them, ADH metabolizes ethanol to acetaldehyde, and ALDH metabolizes acetaldehyde to acetic acid. [0003] There are three types of ADH, ADH1, ADH2, and ADH3, and ADH2*1 (WT), ADH2*2, ADH2*3 are present in the genetic polymorphisms related to the ADH2 gene phenotype (Non-Patent Document 1). The frequency among the Japanese is *1 / *1: 8.4%, *1 / *2: 34.9%, *2 / *2: 56.7%, almost non-existent*3 (Non-Patent Document 2). [0004] The frequency of *1 (WT) and *2 (Non-Patent Document 1) among the genetic polymorphisms related to ALDH2 gene phenotype among Japanese is *1 / *1: 52.8%, *1 / *2: 40.9 %, *2 / *2: 6.3% (Non-Pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
CPCC12Q2527/101C12Q1/6827C12Q1/6837
Inventor 平井光春井口亚希
Owner ARKRAY INC
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