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Primer and method for detecting ADH1B gene*2 polymorphism

A polymorphism and gene technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, can solve the problems of insufficient accuracy of ALDH2 gene*2 and ADH1B gene*2 polymorphism, etc. To achieve the effect of good specificity, high sensitivity and high detection efficiency

Inactive Publication Date: 2016-04-20
GUANGZHOU KINGMED HEALTH CONSULTING CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent application 201510085568.2 discloses a primer set and sequencing primers for detecting the genotype of the single nucleoside polymorphism R49H of the ADH1B gene and the single nucleoside polymorphism E487K of the ALDH2 gene, but it is used to detect ALDH2 Insufficient accuracy of polymorphisms of gene *2 and ADH1B gene *2
The prior art lacks a primer and method for detecting ADH1B gene *2 polymorphism with high detection specificity and sensitivity, good accuracy and precision

Method used

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  • Primer and method for detecting ADH1B gene*2 polymorphism
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  • Primer and method for detecting ADH1B gene*2 polymorphism

Examples

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Effect test

Embodiment 1

[0022] Example 1 Primer

[0023] The inventor designed a large number of primers for the gene polymorphism site of ADH1B gene *2, and selected primers with good specificity through optimization and comparison of primer reaction conditions. The primers provided by the present invention are shown in Table 1. All primer sequences provided by the present invention are compared through UCSC database, and there is no known SNP site.

[0024]

Embodiment 2

[0025] The specificity of embodiment two primers

[0026] The ADH1B gene*2 primers provided by the present invention were blasted in UCSC, and the results were as follows: the amplified fragment of ADH1B gene*2 was located at chr4:100239170+100239459, with a length of 290bp. The amplified fragments and genomic locations of ADH1B gene*2 specific primers are as follows figure 1 As shown, the amplified fragments of the primers covered the corresponding detection site ADH1B gene*2, and there were no other homologous genes.

[0027] The PCR amplification primers in Table 1 were used to amplify and Sanger sequence the DNA of the detection sample respectively, and some sequencing results are as follows figure 2 shown. Sequencing results showed that the fragments amplified by the primers matched the reference sequence of the ADH1B gene*2 gene. The ADH1B gene*2 gene reference sequence number is ADH1BNC_000004.12, NM_000668.5.

[0028] Using the SNaPshotPCR primers in Table 1, the ...

Embodiment 3

[0029] Example 3 Detection of ADH1B gene *2 polymorphism

[0030] 1. Extract DNA samples from EDTA anticoagulated peripheral blood. The extraction method refers to the instructions of TIANampBloodDNAKit (purchased from Tiagen, product number DP318). Dilute the DNA samples to 100ng / μL and set aside.

[0031] 2. Multiplex PCR amplification

[0032] (1) PCR amplification using Q5 ? Hot-start ultra-fidelity 2XMasterMix (purchased from NEB Company, catalog number M0494L), using the extracted sample DNA as a template, the reaction system is shown in Table 2, and the concentration of PCR amplification primers is 5 pmol / uL.

[0033]

[0034] (2) Multiplex PCR amplification reaction conditions include: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 29 cycles, and a final extension at 72°C for 5 min, followed by incubation at 25°C.

[0035] (3) After the PCR amplification is completed, take 1.5 μL of the P...

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Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a primer and a method for detecting ADH1B gene*2 polymorphism. The primer for detecting the ADH1B gene*2 polymorphism comprises a PCR amplification primer and an SNaPshot PCR primer. According to the primer and the method for detecting the ADH1B gene*2 polymorphism, specific detection of the ADH1B gene*2 polymorphism can be realized, the detection specificity and sensitivity are high, accuracy and precision are good, the primer and the method can be used for clinically detecting the ADH1B gene*2 polymorphism, and a detection result can be used for guiding people to healthily drink alcohol and reducing the occurrence of alcoholism.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a primer and a method for detecting ADH1B gene *2 polymorphism. Background technique [0002] Alcohol dehydrogenase (encoded by ADH1B gene) is one of the key enzymes in alcohol metabolism. Its mutation can affect alcohol metabolism and cause the accumulation of harmful substances such as acetaldehyde, which affects health. Studies have shown that there are 3 alleles at the ADH1B gene locus, encoding 3 subunits, namely β1 (ADH1B*1), β2 (ADH1B*2) and β3 (ADH1B*3). Alcohol dehydrogenase consisting of the β2 polypeptide encoded by the ADH1B*2 allele is highly active. ADH1B gene *2 polymorphism (NM_000668.5:c.143A>G, p.His48Arg) can lead to a great increase in the activity of alcohol dehydrogenase, and the metabolism of ethanol to acetaldehyde is faster, which promotes the accumulation of acetaldehyde in the body . Therefore, the ADH1B gene *2 gene polymorphism can ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113C12Q2565/125
Inventor 燕启江赵薇薇胡昌明梁耀铭于世辉郭周萍
Owner GUANGZHOU KINGMED HEALTH CONSULTING CO LTD
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