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

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

Example Embodiment

[0028] Example 1: Primer.

[0029] A large number of primers were designed for the genetic polymorphisms of ALDH2 gene SNP locus rs671 and ADH1B gene SNP locus rs1229984. Through the optimization and comparison of primer reaction conditions, the specificity, no cross-reaction and PCR were selected. Primers with very close reaction conditions.

[0030] Primer pair to amplify ALDH2 gene sequence fragment:

[0031] F1 (SEQIDNO.: 1): 5'-CGGGGAGTGGCCGGGAGTTGGGC-3', 5'for biotin labeling

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

[0033] Primer pair for amplification of ADH1B gene sequence fragment:

[0034] F2 (SEQIDNO.: 3): 5'-CAGCTTCTCTTTATTCTGTAGAT-3', 5'for biotin labeling

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

[0036] Pyrosequencing primers at SNP site rs671 of ALDH2 gene:

[0037] SEQIDNO: 5: 5'-ACTCACAGTTTTCACTT-3';

[0038] Pyrosequencing primers at SNP site rs1229984 of ADH1B gene:

[0039] SEQIDNO: 6: 5'-ACCACGTGGTCATCTGT-3'.

Example Embodiment

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

[0041] 1. Extract the DNA sample of EDTA anticoagulated whole blood: The extraction method refers to the instruction of TIANamp Blood DNA Kit (purchased from Tiagen, item number DP318), and the DNA sample is diluted to 50ng / μL for use;

[0042] 2. Multiplex PCR amplification:

[0043] (1) Prepare PCR amplification primer mixture, the primer concentration ratio of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984 is 1:1, the final concentration of the primer mixture is 10μmol / L, shake and mix well and microcentrifuge. PCR amplification adopts TaKaRa PCR Amplification Kit (purchased from TaKaRa company, article number R011), using the extracted sample DNA as a template, and the reaction system is shown in Table 1;

[0044] Table 1 Multiplex PCR amplification system

[0045]

[0046]

[0047] (2) Multiple PCR amplification reaction con...

Example Embodiment

[0052] Example 3: Clinical sample detection.

[0053] The primers and methods for detecting alcohol metabolism genes by the pyrosequencing combined with sequencing method of the present invention were used to simultaneously detect the polymorphisms of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984 on 20 clinical samples, and the detection method was implemented according to the implementation The steps in Example 2 are implemented. At the same time, common pyrosequencing method was used to detect the polymorphisms of ALDH2 gene SNP site rs671 and ADH1B gene SNP site rs1229984, and the test 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] The detection results obtained by using the pyrosequencing combined sequencing method to detect the alcohol metabolism gene primers and methods provided by the present invention are consistent with those obta...

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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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