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Primer for detecting alcohol metabolizing genes by aid of pyrosequencing joint sequencing methods and application of primer

A technology of pyrosequencing and sequencing primers, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as the reduction of acetaldehyde dehydrogenase enzyme activity and the impact on the efficiency of angina pectoris, etc. Achieve the effect of good accuracy, short detection period and high throughput

Active Publication Date: 2017-07-28
石家庄迪安医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ALDH2 gene SNP site rs671 polymorphism leads to a significant decrease in the activity of acetaldehyde dehydrogenase, thereby affecting the effectiveness of nitroglycerin in the treatment of angina pectoris

Method used

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  • Primer for detecting alcohol metabolizing genes by aid of pyrosequencing joint sequencing methods and application of primer
  • Primer for detecting alcohol metabolizing genes by aid of pyrosequencing joint sequencing methods and application of primer
  • Primer for detecting alcohol metabolizing genes by aid of pyrosequencing joint sequencing methods and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Primers.

[0029] A large number of primers were designed for the gene polymorphism sites of the SNP site rs671 of the ALDH2 gene and the SNP site rs1229984 of the ADH1B gene. Primers with very close reaction conditions.

[0030] Primer pairs for amplifying ALDH2 gene sequence fragments:

[0031] F1 (SEQ ID NO.: 1): 5'-CGGGGAGTGGCCGGGAGTTGGGC-3', 5' for biotin labeling

[0032] R1 (SEQ ID NO.: 2): 5'-CCTCAAGCCCCAACAGGCCCTGA-3';

[0033] Primer pairs for amplifying ADH1B gene sequence fragments:

[0034] F2 (SEQ ID NO.: 3): 5'-CAGCTTCTCTTTATTCTGTAGAT-3', 5' for biotin labeling

[0035] R2 (SEQ ID NO.: 4): 5'-CCTAAAATCACAGGAAGGGGGG-3';

[0036] Pyrosequencing primers for ALDH2 gene SNP site rs671:

[0037] SEQ ID NO: 5: 5'-ACTCACAGTTTTTCACTT-3';

[0038] Pyrosequencing primers for ADH1B gene SNP site rs1229984:

[0039] SEQ ID NO: 6: 5'-ACCACGTGGTCATCTGT-3'.

Embodiment 2

[0040] Example 2: Simultaneous detection of the polymorphisms of the SNP site rs671 of the ALDH2 gene and the SNP site rs1229984 of the ADH1B gene.

[0041] 1. Extract DNA samples from EDTA anticoagulated whole blood: the extraction method refers to the instructions of TIANamp Blood DNA Kit (purchased from Tiagen, product number DP318), dilute the DNA samples to 50ng / μL, and set aside;

[0042] 2. Multiplex PCR amplification:

[0043](1) Prepare a primer mixture for PCR amplification. The primer concentration ratio of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984 is 1:1, and the final concentration of primer mixture is 10 μmol / L. Shake, mix and microcentrifuge. TaKaRa PCR Amplification Kit (purchased from TaKaRa Company, Cat. No. R011) was used for PCR amplification, and the extracted sample DNA was used as a template. The reaction system is shown in Table 1;

[0044] Table 1 Multiplex PCR amplification system

[0045]

[0046]

[0047] (2) Multiple PCR a...

Embodiment 3

[0052] Example 3: Detection of clinical samples.

[0053] The primers and method for detecting alcohol metabolism genes by pyrosequencing combined sequencing method described in the present invention were used to simultaneously detect polymorphisms of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984 polymorphisms in 20 cases of clinical samples, and the detection method was implemented according to The steps in Example 2 were carried out. At the same time, the polymorphisms of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984 were detected by ordinary pyrosequencing method, and the detection results were compared with the combined detection method. The comparison results are shown in Table 1:

[0054] Table 1 Comparison table of sample test results

[0055]

[0056] Using the pyrosequencing combined sequencing method provided by the present invention to detect alcohol metabolism gene primers and methods, the detection results obtained by analyzing 20 ...

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Abstract

The invention discloses a primer for detecting alcohol metabolizing genes by the aid of pyrosequencing joint sequencing methods, and belongs to the technical field of biological detection. The primer comprises amplification primers shown as SEQ ID NO.1-4 and sequencing primers shown as SEQ ID NO.5-6. Biotin labeling is carried out by 5' ends of the primers shown as SEQ ID NO.1 and SEQ ID NO.3. The invention further discloses application of the primer to simultaneously detecting the polymorphism of SNP (single nucleotide polymorphism) loci rs671 of ALDH2 (acetaldehyde dehydrogenase 2) genes and SNP loci rs1229984 of ADH1B (alcohol dehydrogenase 1B) genes. The primer is high in detection throughput as compared with the traditional ordinary pyrosequencing with only single-SNP polymorphism detection capacity during sequencing reaction in each procedure, the detection time can be effectively shortened, and labor and the cost can be effectively reduced. Besides, the primer and the application have the advantages of accurate detection results, good specificity, high sensitivity, short detection cycle, simplicity in operation and capability of meeting clinical examination requirements.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method and primers for detecting alcohol metabolism genes by pyrosequencing combined sequencing. Background technique [0002] The acetaldehyde dehydrogenase encoded by the ALDH2 gene and the alcohol dehydrogenase encoded by the ADH1B gene are the key enzymes in the process of alcohol metabolism. The substance is further converted into harmless acetic acid (the component of vinegar) under the catalysis of acetaldehyde dehydrogenase. The polymorphism of the coding gene causes the change of the catalytic ability of the enzyme, which will affect the metabolism of alcohol and cause the accumulation of harmful substances such as acetaldehyde, which will affect the health of the body. The G1510A polymorphism (rs671) in the ALDH2 gene will cause the glutamic acid at position 487 of the amino acid sequence to be replaced by lysine (ie, Glu487Lys), and the wild...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/32C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/301C12Q2535/122
Inventor 任绪义张锋曹子豪
Owner 石家庄迪安医学检验实验室有限公司
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