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Probe, primer and kit for detecting polymorphism of ADRB1 gene

A gene polymorphism and kit technology, applied in the field of molecular biology, can solve the problems of high cost, unsuitable detection, time-consuming and laborious, etc., and achieve the effect of simple operation, rapid screening, and improved curative effect

Inactive Publication Date: 2021-02-02
上海利康精准医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe, primer and kit for detecting polymorphism of ADRB1 gene
  • Probe, primer and kit for detecting polymorphism of ADRB1 gene
  • Probe, primer and kit for detecting polymorphism of ADRB1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: probe and primer design

[0028] The present invention designs probes and primer sequences for the ADRB1 SNP site. The specific principle is to use the conformational change of the fluorescent probe and the target sequence after hybridization to release the fluorescent dye, and judge the genotyping results according to the peak diagrams and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined together, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher The extinguishing groups are separated from each other, and the fluorescent signal can be detected.

[0029] Design probes and primers so that when the template does not exist at the annealing temperature, the probe is in a stem-loop state, including the loop sequence and the reverse-complementary stem sequen...

Embodiment 2

[0036] Embodiment 2: Detect different genotype standard products

[0037] 1. Use the plasmid ADRB1 to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the rs1801253 site of the target gene (the source of the plasmid, and the synthesis of the plasmid containing the target gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The accuracy of the sequence was determined by sanger sequencing. The genotype of the wild-type standard plasmid rs1801253 was GG;

[0038] 2. Using the probes and primers in Example 1.

[0039] 3. PCR reaction system:

[0040] 1) Add 7.5ul of PCR Mix, 0.5uM of primer 1 solution and 0.5uM of primer 2 solution, and 0.1uM of probe into each PCR reaction well, and then add the wild-type standard plasmid into 3 different PCR reaction wells respectively 2ul each of DNA, mutant standard plasmid DNA, mixed DNA (wild type standard plasmid and mutant standard plasmid are mixed at a ratio of 1:1), make up 15ul ...

Embodiment 3

[0043] Embodiment 3: detection method performance analysis experiment

[0044] 1. Precision experiment

[0045] The wild-type standard plasmid DNA and the mixed-type DNA (rs1801253 plasmid source, and the synthesis of the plasmid containing the target gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The wild-type standard plasmid and mutant standard plasmid were according to 1: 1 proportion mixed) each part, detect 3 times every day, detect continuously 5 days, the amplification reaction program adopts the method in embodiment 2, the result sees figure 2 . It shows that the fluorescent PCR amplification reaction of the present invention has good repeatability (the coincidence rate is greater than 95%, and the coefficient of variation CV of the Ct value of the detection result is less than 5%).

[0046] 2. Conformity rate experiment

[0047] Select 20 cases of DNA samples from healthy volunteers in Shanghai, apply the method of Example 2 to detect the rs1...

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Abstract

The invention provides a probe for detecting the polymorphism of an ADRB1 gene. The sequence of the probe is shown as SEQ ID NO.: 1, or the two ends of the sequence of SEQ ID NO.: 1 are modified by groups. The invention also provides a primer for detecting the polymorphism of the ADRB1 gene. The sequences of the primer are shown as SEQ ID NO.: 2 and SEQ ID NO.: 3. The probe and primer for detecting the polymorphism of the ADRB1 gene have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when being used for detecting the polymorphism of theADRB1 gene.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting ADRB1 gene polymorphisms. Background technique [0002] β-adrenergic receptor (β-adrenergic receptor) is a subfamily of adrenergic receptors, belonging to the G protein-coupled receptor superfamily, including β 1 , β 2 and beta 3 Three different subtypes. This type of receptor regulates intracellular cAMP and L-type Ca by coupling with Gs protein 2+ The opening frequency of the channel is the target of β-agonists and β-blockers. beta 1 Polymorphisms in the receptor-encoding gene ADRB1 can affect the efficacy of beta-blockers such as metoprolol. The ADRB1 Gly389Arg (rs1801253) polymorphism leads to two types of receptors at the site Arg389 and Gly389, and the coupling efficiency of the Arg389 type receptor to G protein is higher than that of the Gly389 type receptor. Arg389 homozygous hypertensive patients treated with metoprolol have ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6827C12N15/11
CPCC12Q1/6883C12Q1/6827C12Q2600/106C12Q2600/156
Inventor 张奕李吉明王校毛丹丹
Owner 上海利康精准医疗技术有限公司
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