Genotype determination method

A genotype and gene technology, applied in the field of gene genotype, can solve problems such as complicated procedures

Inactive Publication Date: 2012-07-04
EDUCATIONAL CORP MUKOGAWA GAKUIN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in any of the methods disclosed in these Patent Documents 1-3, since procedures such as purification of DNA used in the PCR method are complicated, it is necessary to develop a simpler method.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] (1) Determine genotype by PCR-RFLP method

[0083] Gene polymorphisms of alcohol dehydrogenase ADH1B gene and acetaldehyde dehydrogenase ALDH2 gene were detected for blood (whole blood), saliva (including cells in the oral cavity), and hair roots respectively collected from the test subjects in the following order. For blood and saliva, soak on filter paper (Advantec qualitative filter paper No. 2), let it dry naturally, and collect it as a solid sample to be tested. The usual DNA extraction and purification processes were omitted, and the 1 mm-diameter perforated piece (Panchi piece) of the dry filter paper was directly placed in the PCR reaction solution in the tubular carrier. In addition, for hairy roots, they were directly put into the PCR reaction solution inside the tubular carrier. PCR reaction solution Using the PCR kit KOD FX (manufactured by TOYOBO Co., Ltd.), following the attached protocol, 25 μL of reaction solutions containing the following reagents were...

Embodiment 2

[0172] The same as in Example 1, soak the collected blood into filter paper (Advantec qualitative filter paper No. 2), make it dry naturally, and as a solid sample to be tested, directly put the 1mm-diameter perforated sheet of the dry filter paper into the tubular carrier in the PCR reaction solution. PCR reaction solution Using the PCR kit KODFX (manufactured by TOYOBO Co., Ltd.), following the attached protocol, 25 μL of reaction solutions containing the following reagents were respectively prepared.

[0173] (PCR reaction solution for ADH1B gene)

[0174] ・2×PCR buffer for KOD FX: 10 μL

[0175] dNTPs: 2 μL

[0176] Primer 5 (ADH1B-F) (10 μM): 0.8 μL

[0177] Primer 6 (ADH1B-R) (10 μM): 0.8 μL

[0178] · KOD FX: 0.4 μL

[0179] · DW (distilled water): 6 μL.

[0180] (PCR reaction solution for ALDH2 gene)

[0181] ・2×PCR buffer for KOD FX: 10 μL

[0182] dNTPs: 2 μL

[0183] Primer 7 (ALDH2-F) (10 μM): 0.8 μL

[0184] Primer 8 (ALDH2-R) (10 μM): 0.8 μL

[0185] ·...

Embodiment 3

[0223] Using the following restriction enzyme reaction solution, the amplification products of ADH1B gene and ALDH2 gene obtained in the same manner as in Example 2 were simultaneously subjected to restriction enzyme digestion reaction at 37°C for 1 hour.

[0224] (restriction enzyme reaction solution)

[0225] 10×NEB buffer 4: 2 μL

[0226] Amplified product of ADH1B gene: 3 μL

[0227] Amplified product of ALDH2 gene: 3 μL

[0228] 8×SAM: 2.5 μL

[0229] Msl 1: 0.5 μL

[0230] Acu 1: 0.5 μL

[0231] · DW (distilled water): 8.5 μL.

[0232] The reaction fragments of ADH1B gene and ALDH2 gene were respectively loaded on agarose gel, and the amplified genes were confirmed by electrophoresis. In addition, DW was also used as a control. Figure 5 It is an electrophoresis photograph showing the result of Example 3.

[0233] exist Figure 5 , the bars show the following results.

[0234] Stripe 1: ADH1B*1 / *2, ALDH2*1 / *2

[0235] ·Strip 2: ADH1B*1 / *1, ALDH2*2 / *2

[0236]...

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Abstract

Disclosed is a method for determining a genotype of a gene associated with alcohol susceptibility, which comprises the steps of: bringing a solid test sample containing the gene into direct contact with a solution comprising a buffer and a DNA polymerase and primer DNA for the amplification of the gene to carry out a method selected from a polymerase chain reaction, an LAMP method, a chain substitution amplification method, a reverse transcriptase chain substitution amplification method, a reverse transcriptase polymerase chain reaction, a reverse transcription LAMP method, an amplification method based on a nucleotide sequence, a transcription-mediated amplification method, and a rolling circle-type amplification method, thereby amplifying the gene; and determining the genotype of the gene using the amplified gene. The method enables the determination of the genotype of a gene associated with alcohol susceptibility safely, rapidly and at low cost.

Description

technical field [0001] The present invention relates to a method for judging the genotype of the gene contained in the solid test sample from the solid test sample containing the gene related to ethanol sensitivity (ethanol sensitivity related gene). Background technique [0002] Alcohol dehydrogenase ADH (Alcohol Dehydrogenase) is an enzyme involved in decomposing ethanol into acetaldehyde in the body, and various types are known. Among them, in the gene ADH1B (formerly known as ADH2), there is a genotype ADHB1*1 / *1 called the low-activity type whose ethanol metabolism is slow. (flushing) symptoms, because ethanol is decomposed slowly, so ethanol tends to remain in the body, and it is called a constitution that is prone to alcohol dependence. [0003] Also, acetaldehyde dehydrogenase ALDH (Aldehyde Dehydrogenase) is one of enzymes involved in decomposing acetaldehyde into acetic acid in the body, and various types are known. Among them, among the ALDH2 genes that function...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q2600/156C12Q2600/158C12Q1/6883
Inventor 木下健司
Owner EDUCATIONAL CORP MUKOGAWA GAKUIN
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